Two B3 domain transcriptional repressors prevent sugar-inducible expression of seed maturation genes in Arabidopsis seedlings

Abstract

During development of plant seeds, embryos import nutrients and store massive amounts of reserves. Seed reserves are rapidly degraded and mobilized to support seedling development after germination. HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE 2 (HSI2) of Arabidopsis thaliana is a B3 DNA-binding domain protein that represses the transcription of sugar-inducible reporter gene. Although disruption of HSI2 or HSI2-Like 1 (HSL1) did not affect growth, seeds with disruption of both HSI2 and HSL1 (KK mutant) developed abortive seedlings that stopped growing 7-9 days after imbibition. KK seedlings developed swollen hypocotyls that accumulated seed storage proteins and oil on medium containing sucrose or other metabolizable sugars, and calluses developed from KK seedlings also accumulated seed storage reserves. The expression of seed maturation genes, which include LEAFY COTYLEDON-type master regulators, in KK seedlings depended on the concentration of sucrose, suggesting that sugar controls the expression of seed maturation genes. Our results suggest that HSI2 and HSL1 repress the sugar-inducible expression of the seed maturation program in seedlings and play an essential role in regulating the transition from seed maturation to seedling growth.

Fig. 1.

HSI2 subfamily of B3 domain proteins and phenotypic features of KK seedlings. (A) Amino acid sequences of B3 domain of Arabidopsis B3 domain proteins were aligned by using ClustalW, and a phylogenetic neighbor-joining tree was generated as described previously (10). (B) Schematic structures of HSI2 and HSL1. The locations of the B3 domain and EAR motif are shown. (C) Col-0 and KK seedlings germinated on medium containing 1% sucrose for 3, 5, or 7 days. (D) Cross-sections of hypocotyls of 5-day-old Col-0 and KK seedlings germinated on medium containing 1% sucrose and were stained with toluidine blue. (E) Col-0 and KK seedlings germinated on sucrose-free medium for 5 days. [Scale bars, 0.5 mm (C–E).]

Fig. 2.

Accumulation of seed storage compounds in KK seedlings. (A) Proteins extracted from 4-, 7-, and 9-day-old Col-0 and KK seedlings grown on medium containing 0% or 1% sucrose were analyzed by immunoblotting with anti-12S globulin and anti-oleosin S4 antibodies. Each lane contained 1 μg of protein. Proteins extracted from Col-0 seeds served as a control, and the size of protein molecular mass standards is indicated. (B) Five- and 12-day-old Col-0 and KK seedlings on medium containing 1% sucrose were stained with fat red 7B. (Scale bar, 0.5 mm.) (C) Total lipids were extracted from 4- and 9-day-old Col-0 and KK seedlings grown on medium containing 0% or 1% sucrose, and those from four seedlings were separated by TLC and stained with sulfuric acid. Lipids extracted from four Col-0 seeds served as a control, and 10 μg of glyceryl trilinoleate was used as a standard (Std).

Fig. 3.

Expression of HSI2, HSL1, and seed maturation genes in Col-0 and KK seedlings. Total RNAs were isolated from 4-, 5-, 7-, and 9-day-old Col-0 and KK seedlings grown on medium containing 1% sucrose. The levels of various mRNAs were determined by quantitative real-time RT-PCR using ACTIN2 mRNA as an internal reference. Results represent the average from two independent isolations of RNA ± SD.

Fig. 4.

Effects of the concentration of sucrose in the medium on the expression of various genes in seedlings. Total RNA was isolated from 5-day-old Col-0 and KK seedlings grown on medium containing 0, 10, 30, and 90 mM sucrose. The levels of various mRNAs were determined by quantitative real-time RT-PCR using ACTIN2 mRNA as an internal reference. Results represent the average of data from two independent isolations of RNA ± SD.

Fig. 5.

Endogenous level of ABA in Col-0 and KK seedlings. The ABA content in 7-day-old Col-0 and KK seedlings germinated on medium containing various concentrations of sucrose was determined. Results represent the average from three independent experiments ± SD.

Fig. 6.

Calluses developed from hypocotyls of KK seedlings. (A) Hypocotyls of 7-day-old Col-0 and KK seedling were excised and cultured on medium containing 1% sucrose for 14 days. (Right) The photographs were taken after staining with fat red 7B. (Scale bar, 1 mm.) (B) Proteins were extracted from Col-0 and KK hypocotyls that had been excised and cultured for 14 days (Col explant and KK callus, respectively), callus from Col-0 explants on CIM (CIM callus), and Col-0 seeds (Seed). Equal amounts of proteins (1 mg) were separated by SDS/PAGE and stained with Coomassie brilliant blue (Left) or analyzed by immunoblotting with anti-12S globulin and anti-oleosin S4 antibodies (Right). The size of the protein molecular mass standards is indicated to the left of the gels.

Fig. 7.

Schematic illustration of the role of HSI2, HSL1, and other genes in Arabidopsis seed development.