Arabidopsis Hexokinase-Like1 and Hexokinase1 form a critical node in mediating plant glucose and ethylene responses

Abstract

Arabidopsis (Arabidopsis thaliana) Hexokinase-Like1 (HKL1) lacks glucose (Glc) phosphorylation activity and has been shown to act as a negative regulator of plant growth. Interestingly, the protein has a largely conserved Glc-binding domain, and protein overexpression was shown previously to promote seedling tolerance to exogenous 6% (w/v) Glc. Since these phenotypes occur independently of cellular Glc signaling activities, we have tested whether HKL1 might promote cross talk between the normal antagonists Glc and ethylene. We show that repression by 1-aminocyclopropane-1-carboxylic acid (ACC) of the Glc-dependent developmental arrest of wild-type Arabidopsis seedlings requires the HKL1 protein. We also describe an unusual root hair phenotype associated with growth on high Glc medium that occurs prominently in HKL1 overexpression lines and in glucose insensitive 2-1 (gin2-1), a null mutant of Hexokinase1 (HXK1). Seedlings of these lines produce bulbous root hairs with an enlarged base after transfer from agar plates with normal medium to plates with 6% Glc. Seedling transfer to plates with 2% Glc plus ACC mimics the high-Glc effect in the HKL1 overexpression line but not in gin2-1. A similar ACC-stimulated, bulbous root hair phenotype also was observed in wild-type seedlings transferred to plates with 9% Glc. From transcript expression analyses, we found that HKL1 and HXK1 have differential roles in Glc-dependent repression of some ethylene biosynthesis genes. Since we show by coimmunoprecipitation assays that HKL1 and HXK1 can interact, these two proteins likely form a critical node in Glc signaling that mediates overlapping, but also distinct, cellular responses to Glc and ethylene treatments.

Figure 1.

ACC blocks the Glc-dependent developmental arrest of wild-type seedlings but not of hkl1-1 seedlings. The bars indicate corresponding parental controls for modified lines. Lr-OE = HKL1 overexpression line 52 in the Ler background; gn-OE = HKL1 overexpression line 79 in the gin2-1 background. In the top panel, seedlings were grown 7 d on plates with 6% (w/v) Glc. In the bottom panel, seedlings were grown 7 d on plates with 6% (w/v) Glc + 50 μm ACC. [See online article for color version of this figure.]

Figure 2.

Roots of several genotypes formed abnormal root hairs with basal bulges after seedling transfer to agar plates with 6% (w/v) Glc. At least 10 seedlings of each genotype were grown vertically under constant light (30 μmol m⁻² s⁻¹) and with different conditions, as follows: column 1, seedlings were grown continuously on 0.5% (w/v) Suc plates; column 2, seedlings were grown initially for 7 d on 0.5% (w/v) Suc plates and then transferred for 6 d to 6% (w/v) Glc plates; column 3, seedlings were grown continuously on 2% (w/v) Glc plates; column 4, seedlings were grown initially for 7 d on 2% (w/v) Glc plates and then transferred for 4 d to 6% (w/v) Glc plates; column 5, seedlings were grown initially for 7 d on 2% (w/v) Glc plates and then transferred for 6 d to 6% (w/v) mannitol (Man) plates.

Figure 3.

The abnormal root hair phenotype of Lr-OE occurs also by seedling transfer to plates with ACC. At least 10 seedlings of each genotype were grown vertically under constant light (30 μmol m⁻² s⁻¹) and with different conditions, as follows: column 1, seedlings were grown initially for 7 d on 0.5% (w/v) Suc plates and then transferred for 4 d to 0.5% (w/v) Suc plates with 5 μm ACC; column 2, seedlings were grown initially for 7 d on 2% (w/v) Glc plates and then transferred for 4 d to 2% (w/v) Glc plates with 5 μm ACC; column 3, for comparison and as a control, seedlings were grown as in Figure 2, initially for 7 d on 2% (w/v) Glc plates and then transferred for 4 d to 6% (w/v) Glc plates; column 4, seedlings were grown initially for 7 d on 2% (w/v) Glc plates and then transferred for 4 d to 6% (w/v) Glc plates with 1 μm AVG.

Figure 4.

Seedling root hair growth of ethylene mutants ein2-1 and eto2-1 and wild-type Col on plates with different media. A, Continuous growth of ethylene mutants on 2% (w/v) Glc plates or after seedling transfer to plates with 6% (w/v) Glc with or without 1 μm AVG. B, Wild-type Col seedlings were grown initially for 7 d on 2% (w/v) Glc plates and then transferred for 4 d to plates with 7% Glc, 7% Glc + 50 μm ACC, or 7% mannitol. C, Wild-type Col seedlings were transferred to plates with 8% Glc, 8% Glc + 50 μm ACC, or 8% mannitol. D, Wild-type Col seedlings were transferred to plates with 9% Glc, 9% Glc + 50 μm ACC, or 9% mannitol.

Figure 5.

The influence of Glc treatment on the expression of genes related to ethylene metabolism in HKL1 transgenic lines and mutants. Seedlings were grown initially in liquid MS medium, prior to dark adapting in sugar-free medium, followed by 8 h of treatment under light with or without 2% (w/v) Glc. qRT-PCR was used to determine seedling transcript levels of candidate genes identified previously by transcriptional profiling (Price et al., 2004): ACS7 (for ACC synthase 7; At4g26200), DOG (the homolog to tomato E8 dioxygenase protein; At5g26740), ACO (for ACC oxidase; At1g12010), EIN3 (At3g20770), and EIL (At2g27050). Transcript abundance of TPS8 (for trehalose 6-phosphate synthase 8; At1g70290) was monitored as a positive Glc-repressible control, and UBQ5 (At3g62250) was monitored as an internal control. The bars represent relative fold change relative to the respective control genotypes. Error bars represent se of two independent replicate samples.

Figure 6.

Differentially tagged HXK1 and HKL1 can interact with each other by coimmunoprecipitation assay. Maize mesophyll protoplasts were transfected with the indicated combinations of AtHXK1-HA (approximately 54 kD), AtHKL1-GFP (approximately 82 kD), and/or yeast HXK2-HA (YHK2-HA; approximately 54 kD). Newly made proteins were labeled with [³⁵S]Met and then pulled down using anti-HA antibody (H) or anti-GFP antibody (G). The negative control (Neg Control) was protoplasts transfected with an empty vector and then subjected to pull-down assay with the antibodies.

Figure 7.

Proposed model for the roles of HKL1 and HXK1 in mediating cross talk between Glc and ethylene tissue responses. HKL1 is shown as a repressor of two Glc-dependent growth responses, which themselves are positively mediated by HXK1. HXK1-dependent growth responses might result from transcriptional or posttranscriptional regulation. Ethylene is shown as mediating an antagonistic role through HKL1 on these two Glc-dependent growth responses. Additionally, HKL1 and HXK1 are proposed to modulate, in a Glc-dependent fashion, the expression of either positive or negative effectors of ethylene biosynthesis. SAM, Shoot apical meristem.