Signaling from an altered cell wall to the nucleus mediates sugar-responsive growth and development in Arabidopsis thaliana

Abstract

Sugars such as glucose function as signal molecules that regulate gene expression, growth, and development in plants, animals, and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar response8 (hsr8), which enhances sugar-responsive growth and gene expression. Light-grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark-grown hsr8 seedlings showed glucose-hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, was allelic to the MURUS4 (MUR4) gene involved in arabinose synthesis. Dark-grown mur1 and mur3 seedlings also exhibited similar sugar responses to hsr8/mur4. The sugar-hypersensitive phenotypes of hsr8/mur4, mur1, and mur3 were rescued by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar-responsive phenotypes. Genetic analysis showed that sugar-hypersensitive responses in hsr8 mutants were suppressed by pleiotropic regulatory locus1 (prl1), indicating that nucleus-localized PRL1 is required for enhanced sugar responses in hsr8 mutant plants. Microarray analysis revealed that the expression of many cell wall-related and sugar-responsive genes was altered in mur4-1, and the expression of a significant proportion of these genes was restored to wild-type levels in the mur4-1 prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar-responsive metabolic, growth, and developmental changes.

Figure 1.

Isolation of the hsr8-1 Mutant. (A) Luciferase activity in the hsr8-1 mutant is higher than that in the wild type. A3L3 is the parental line of hsr8-1. hsr8-1com is hsr8-1 transformed with the genomic wild-type HSR8 sequence. (B) Luciferase levels were measured in 9-d-old seedlings of the A3L3 parental line, hsr8-1, and hsr8-1com grown on Murashige and Skoog (MS) medium containing 1% glucose. Seedlings were sprayed with luciferin and analyzed luminometrically. Error bars represent se (n > 60). (C) RT-PCR analysis of transcript levels in the hsr8-1 mutant. RT-PCR was performed on first-strand cDNA made from 9-d-old seedlings grown in constant light on medium containing 1% glucose. cDNA was standardized by reference to an actin standard.

Figure 2.

Glucose-Hypersensitive Dark Development in the hsr8-1 Mutant. (A) to (C) Dark development of Col-0 seedlings grown on medium containing 0.05% glucose (A), 0.5% glucose (B), and 1% glucose (C) for 21 d. (D) to (F) Fourteen-day-old dark-grown seedlings of the wild type (right), hsr8-1 (middle), and hsr8-1com (left) grown on MS medium (E) and MS medium containing 1% glucose (D) and 1% mannitol (F). (G) to (I) The leaf primordium of 8-d-old dark-grown seedlings of A3L3 grown on MS medium (G) and MS medium containing 0.5% glucose (H) and 1% glucose (I). (J) to (L) The leaf primordium of 8-d-old dark-grown hsr8-1 seedlings on MS medium with no glucose (J), 0.5% glucose (K), and 1% glucose (L). Bars = 1 mm in (A) to (C), 0.5 cm in (D), 0.2 cm in (E) and (F), and 0.1 μm in (G) to (L).

Figure 3.

Glucose-Hypersensitive Cell Elongation in the hsr8-1 Mutant. (A) Hypocotyl lengths of 14-d-old dark-grown seedlings of A3L3 and hsr8-1 grown on MS medium supplemented with increasing glucose concentrations. Error bars represent sd (n > 30). (B) and (C) Scanning electron microscope images of 4-d-old dark-grown hypocotyls of the parental line A3L3 (B) and hsr8-1 (C) grown on MS medium supplemented with 1% (w/v) glucose. Bars = 100 μm. (D) Hypocotyl diameters of 4-d-old dark-grown seedlings of A3L3 and hsr8-1 grown on MS medium supplemented with 1% (w/v) glucose. Error bars represent sd (n > 10). (E) Hypocotyl elongation in response to ACC treatment. Hypocotyl lengths of 14-d-old dark-grown seedlings of A3L3, hsr8-1, and ein2 mutants grown on MS medium supplemented with 1% glucose in the presence and absence of 10 μM ACC. Error bars represent sd (n > 30).

Figure 4.

Map-Based Cloning and Expression Patterns of the HSR8 Gene. (A) Fine-mapping of the HSR8 locus. The HSR8 locus was mapped to chromosome 1 (Chr1) between markers F1K23 and F17F8. The HSR8 locus was further refined to a 48-kb genomic DNA region between CAPS markers T5I8-908 and T5I8-740 and cosegregated with dCAPS marker At1g30620dCAPS1. The numerals at bottom indicate the number of recombinants identified from F2 plants. (B) HSR8 gene structure, showing the mutated sites of the two hsr8 alleles. The start codon (ATG) and the stop codon (TGA) are indicated. Closed boxes indicate the coding sequences, and lines between boxes indicate introns. The mutation site in hsr8-1 and the T-DNA insertion site in hsr8-2 also are shown. (C) The mutation in hsr8-1 was measured with the dCAPS marker At1g30620dCAPS1. (D) RT-PCR analysis of HSR8/MUR4 gene expression. Total RNA was isolated from roots, stems, leaves, and flowers. (E) Expression of HSR8 in response to glucose after 6 h of treatment. (F) to (J) Histochemical analysis of GUS activity in a HSR8_pro:GUS seedling (F), hypocotyl and shoot apices (G), a primary root (H), a lateral root (I), and a dark-grown seedling (J).

Figure 5.

Dark Development Phenotypes of Cell Wall Biosynthetic Mutants. (A) Dark development phenotypes of 14-d-old wild-type Col-0, mur4, and hsr8 alleles grown on MS medium supplemented with 1% glucose. All hsr8 alleles exhibit increased dark development compared with the wild type. (B) Dark development phenotypes of 14-d-old wild-type Col-0 and hsr8 alleles grown on MS medium supplemented with 1% glucose and 30 mM l-arabinose. The increased dark development phenotypes of hsr8 mutants in response to glucose were restored to wild-type levels by exogenous l-arabinose. (C) Hypocotyl lengths of 14-d-old dark-grown seedlings of the wild type and hsr8 mutants grown on MS medium supplemented with 1% glucose. (D) Hypocotyl lengths of 14-d-old dark-grown seedlings of the wild type and hsr8 mutants grown on MS medium supplemented with 1% glucose and 30 mM l-arabinose. The reduced hypocotyl elongation of hsr8 mutants was rescued by l-arabinose. (E) Dark development phenotypes of 14-d-old wild type Col-0, hsr8-1, mur3-1, and mur1-1 alleles grown on MS medium supplemented with 1% glucose (left) or with 1% glucose + 2 mM boric acid (right). Dark development and hypocotyl length of hsr8-1, mur4-1, mur3-1, and mur1-1 were restored to wild-type levels (see [I]). (F) and (G) Dark development phenotypes of 14-d-old wild type Col-0 (F) and arad1-2 (G) alleles grown on MS medium supplemented with 1% glucose. (H) Hypocotyl lengths of 14-d-old dark-grown seedlings of Col-0, mur1-1, and mur3-1 grown on MS medium supplemented with different glucose concentrations. (I) Hypocotyl lengths of 14-d-old dark-grown seedlings of Col-0, mur1-1, and mur3-1 grown on MS medium supplemented with 1% glucose + 2 mM boric acid. Error bars represent sd (n > 30). Bars = 0.5 mm in (E) and 1 mm in (A), (B), (F), and (G).

Figure 6.

The Glucose-Responsive Phenotypes of Dark-Grown mur4-1 Seedlings Are Suppressed by prl1. (A) to (D) Fourteen-day-old dark-grown seedlings of the wild type (A), mur4-1 (B), prl1 (C), and mur4-1 prl1 (D) grown on MS medium supplemented with 1% glucose. (E) Hypocotyl lengths of 14-d-old dark-grown seedlings of the wild type, prl1, mur4-1, and mur4-1 prl1 grown on MS medium supplemented with different glucose concentrations. Error bars represent sd (n > 30). (F) and (G) Constant light–grown seedlings of the wild type, mur4-1, prl1, and mur4-1 prl1 grown on MS medium supplemented with 1% glucose (F) and 3% glucose (G). (H) RT-PCR analysis of transcript levels in Col-0, mur4-1, prl1, and mur4-1 prl1. RT-PCR was performed on first-strand cDNA made from 9-d-old seedlings grown in constant light on medium containing 1% glucose. cDNA was standardized by reference to an actin standard. Bars = 2 mm in (A) to (D) and 1 cm in (F) and (G).

Figure 7.

Gene Expression Profiles in mur4-1, prl1, and mur4-1 prl1. (A) Upregulated genes in mur4-1 were classified into six clusters according to their expression profiles in the wild type, mur4-1, prl1, and mur4-1 prl1. Cluster numbers and mutant alleles are indicated as follows: C, Col-0; m, mur4-1; p, prl1; mp, mur4-1 prl1. (B) Downregulated genes in mur4-1 were classified into six clusters according to their expression profiles in the wild type, mur4-1, prl1, and mur4-1 prl1. Cluster numbers and mutant alleles are indicated as in (A). (C) Model of cell wall signaling based on mutant and gene expression analysis.