TRANSTHYRETIN-LIKE and BYPASS1-LIKE co-regulate growth and cold tolerance in Arabidopsis

Abstract

Background: Cold stress inhibits normal physiological metabolism in plants, thereby seriously affecting plant development. Meanwhile, plants also actively adjust their metabolism and development to adapt to changing environments. Several cold tolerance regulators have been found to participate in the regulation of plant development. Previously, we reported that BYPASS1-LIKE (B1L), a DUF793 family protein, participates in the regulation of cold tolerance, at least partly through stabilizing C-REPEAT BINDING FACTORS (CBFs). In this study, we found that B1L interacts with TRANSTHYRETIN-LIKE (TTL) protein, which is involved in brassinosteroid (BR)-mediated plant growth and catalyses the synthesis of S-allantoin, and both proteins participate in modulating plant growth and cold tolerance.

Results: The results obtained with yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that B1L directly interacted with TTL. Similar to the ttl-1 and ttl-2 mutants, the b1l mutant displayed a longer hypocotyl and greater fresh weight than wild type, whereas B1L-overexpressing lines exhibited a shorter hypocotyl and reduced fresh weight. Moreover, ttl-1 displayed freezing tolerance to cold treatment compared with WT, whereas the b1l mutant and TTL-overexpressing lines were freezing-sensitive. The b1l ttl double mutant had a developmental phenotype and freezing tolerance that were highly similar to those of ttl-1 compared to b1l, indicating that TTL is important for B1L function. Although low concentrations of brassinolide (0.1 or 1 nM) displayed similarly promoted hypocotyl elongation of WT and b1l under normal temperature, it showed less effect to the hypocotyl elongation of b1l than to that of WT under cold conditions. In addition, the b1l mutant also contained less amount of allantoin than Col-0.

Conclusion: Our results indicate that B1L and TTL co-regulate development and cold tolerance in Arabidopsis, and BR and allantoin may participate in these processes through B1L and TTL.

Keywords: Arabidopsis; BYPASS1-LIKE; Cold tolerance; Plant growth; TRANSTHYRETIN-LIKE.

Fig. 1

B1L interacts with TTL. a Y2H analysis of the interaction between B1L and TTL. Each yeast clone containing TTL-pGADT7 (TTL-AD) or pGADT7 (AD) together with B1L-pGBKT7-B1L (B1L-BD) or pGBKT7 (BD) was grown on transformation selection (SD:/−W-L) or interaction selection (SD:/−W-L-H-A) plates. Dilution of the inoculation is shown at the top of the picture. Yeast growth on SD:/−L-T-H-A indicates a positive protein-protein interaction. b BiFC analysis in N. benthamiana showing the interaction between B1L and TTL. The N-terminal half of yellow fluorescent protein (YFP^N) was fused to B1L (B1L-YFP^N) and the C-terminal half of yellow fluorescent protein (YFP^C) was fused to TTL (TTL-YFP^C). The constructs were co-transformed into tobacco leaf cells, and fluorescence images were obtained by confocal microscopy. Panels from left to right show signals of yellow fluorescence, a bright-field image, and an overlay of yellow fluorescence and bright-field image images. Bar = 50 μm

Fig. 2

TTL restrains Arabidopsis seedling development. a Phenotypic comparison between 7-day-old ttl-1, TTL-OE and WT seedlings. Bar = 1 cm. b Fresh weight (mg) of ttl-1, TTL-OE, and WT seedlings showed in (a). c Primary root length of ttl-1, TTL-OE, and WT seedlings showed in (a). d Hypocotyl growth of 7-day-old ttl-1, TTL-OE, and WT seedlings in the dark conditions. All seedlings were grown on MS plates at 22 °C in a 16 h:8 h light:dark cycle (a, b, and c) or for 24 h in the dark (d). Data in (b, c, and d) are expressed as the mean value ± SEM (n = 24). Asterisks indicate significant differences (*p < 0.05) from the wild type

Fig. 3

B1L inhibits Arabidopsis seedling development. a Phenotypic comparison between 7-day-old b1l, B1L-OE and WT seedlings. Bar = 1 cm. b Fresh weight (mg) of b1l, B1L-OE, and WT seedlings showed in (a). c Primary root length of b1l, B1L-OE, and WT seedlings showed in (a). d Hypocotyl growth of 7-day-old b1l, B1L-OE, and WT seedlings in the dark conditions. All seedlings were grown on MS plates at 22 °C in a 16 h:8 h light:dark cycle (a, b, and c) or for 24 h in the dark (d). Data in (b, c, and d) are expressed as the mean value ± SEM (n = 24). Asterisks indicate significant differences (*p < 0.05) from the wild type

Fig. 4

ttl-1 and b1l ttl mutants were both more tolerant to freezing than WT under non-acclimation conditions. Freezing tolerance (a, c) and survival rates (b, d) of 3-week-old WT, b1l, ttl-1, and b1l ttl under non-acclimated (NA) or cold-acclimated (CA) conditions. Seven-day-old seedlings grown on MS plates were transplanted to soil and grown at 22 °C for 2 weeks under long day conditions (light:dark, 16 h:8 h). The plants were then treated at − 10 °C for 1 h (NA) or were pretreated at 4 °C for 3 days and then treated at − 10 °C for 6 h (CA). For each line, the survival rate assay was performed with approximately 64 plants and then scored 5 days later. The data are shown as the means of four independent biological replicates ± SEM. Asterisks indicate significant differences (*p < 0.05) from the wild type

Fig. 5

BR restrained the promoted development of the ttl-1 mutant, but not the b1l mutant, under normal conditions. a Primary root length of 7-day-old wild type, ttl-1, and ttl-2 seedlings after treatment with brassinolide, a familiar compound used to analyse the function of BRs in plant growth. b Primary root length of 7-day-old wild type, b1l, and B1L-OE seedlings after treatment with brassinolide. Seedlings in (a) and (b) were germinated and grown on MS plates containing increased concentrations of brassinolide at 22 °C in a 16 h:8 h light:dark cycle. c Hypocotyl growth of 7-day-old wild type and b1l seedlings with brassinolide treatment in the dark. The different concentrations of brassinolide (0.1, 1, 10, 100 nM groups) in panels a, b and c were all dissolved in 80% ethanol. After filter sterilization, they were added to MS plates [1:10000 (v/v)]. The MS plates with 80% ethanol [1:10000 (v/v)] were used as controls (0 nM group). d Hypocotyl growth of 7-day-old wild type and b1l seedlings with brassinazole treatment in the dark. Brassinazole is an inhibitor of BR biosynthesis. The different concentrations of brassinazole (0.025, 0.05, 0.1, 0.2 μM groups) were all dissolved in DMSO and then added to MS plates [1:1000 (v/v)]. MS plates with DMSO [1:1000 (v/v)] were used as a control (0 μM group). Seedlings in (c) and (d) were germinated and grown on MS plates containing increased concentrations of brassinolide or brassinazole at 22 °C grown in the dark condition. Each data point in panels (a, b, c, and d) represents the mean value ± SEM (n = 24). Asterisks indicate significant differences (*p < 0.05) compared with the wild type at each brassinolide or brassinazole concentration

Fig. 6

ttl1–1 and b1l exhibited promoted seedling development under cold conditions, and BR restrained the promoted hypocotyl length of b1l mutants under cold conditions. a Hypocotyl growth of 2-week-old wild type and ttl-1 seedlings under cold conditions in the dark. b Hypocotyl growth of 2-week-old wild type and ttl-1 seedlings under cold conditions in the dark. Seedlings in (a) and (b) were germinated and grown on MS plates at 12 °C for 24 h in the dark. Each data point is expressed as the mean value ± SEM (n = 24). Asterisks indicate significant differences (*p < 0.05) compared with the wild type. c Hypocotyl growth of 2-week-old wild type and b1l seedlings with BR treatment in the dark under cold conditions. Seedlings were germinated and grown on MS plates containing increased concentrations of brassinazole at 12 °C in the dark conditions. Each data point represents the mean value ± SEM (n = 24). Asterisks indicate significant differences (*p < 0.05) compared with the wild type at each brassinolide concentration

Fig. 7

Endogenous allantoin levels in 2-week-old wild type, b1l, ttl-1, and b1l ttl seedlings. Data represent the means ± SEM from three independent experiments. Asterisks indicate significant differences (*p < 0.05) from the wild type