Signaling role of fructose mediated by FINS1/FBP in Arabidopsis thaliana

Abstract

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1-dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1-dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination.

Figure 1. Differential seedling response to fructose signaling.

(A) WT (Ler), gin2, and HXK1 WT, S177A, and G104D-complemented gin2 showed seedling developmental arrest phenotypes on MS agar media containing 6% fructose. The seedlings were grown for 5 d under constant light. Scale bar, 5mm. (B) Unlike WT (Col), gin1 and ctr1 seedlings showed resistance to fructose. (C) CAB2 repression in the presence of high levels of glucose was de-repressed in gin2 seedlings, but not in the presence of high levels of fructose. Gene expression was measured in 5-d-old seedlings grown on MS agar media containing 6% glucose, fructose, or mannitol. (D) CAB2 expression was repressed by both glucose and fructose in WT, but not in gin1 and ctr1 seedlings. Values were normalized based on those obtained from seedlings grown on mannitol, and the means of triplicate measurements are shown with error bars. The experiments were repeated twice with consistent results.

Figure 2. Function and localization of FBP in response to fructose.

(A) A simplified schematic diagram of the sucrose biosynthesis pathway. The fructose metabolic pathway includes fructokinase1 (FRK1), phosphofructokinase1 (PFK1), and putative fructose-1,6-bisphopatase (FBP). (B) FBP suppressed CAB2-fLUC activity. Leaf mesophyll protoplasts were cotransfected with CAB2-fLUC together with PFK1, FRK1, FBP (FBP_3 or FBP_4), or SSM (FBP^S126AS127A). UBQ10-rLUC was cotransfected as a transfection control. An empty vector was used as a control for effectors. Protein expression was analyzed by protein blotting using an anti-HA antibody. (C) FBP was localized to both the nucleus and cytoplasm. Leaf mesophyll protoplasts were transfected with FBP-GFP and then incubated for 5 h or 18 h. GFP-only and EIN3-GFP constructs were transfected and examined as control proteins localized to the cytosol and nucleus, respectively. GFP was observed under a fluorescence microscope (200× magnification). Cell images were also taken under white light as a control.

Figure 3. FINS1/FBP in fructose signaling.

(A) Molecular analysis of fins1. T-DNA insertion sites and primer (LP, RP, and LB) locations are indicated. Expression of FINS1 was shown by reverse transcription-PCR with a gene-specific primer set (Table S1). UBQ10 served as an internal control. (B) The fins1 and gin2 mutants showed different sensitivities to 6% glucose (Glc) and fructose (Fru). The seedlings were grown for 5 d under a 16 h photoperiod. Scale bar, 5 mm. (C) FINS1 and HXK1 expression suppressed CAB2-fLUC activity. Protoplasts isolated from fins1 seedlings were cotransfected with CAB2-fLUC and HXK1, FINS1/FBP_3, or FINS1/FBP_4. UBQ10-rLUC was used as a transfection control. An empty vector served as a treatment control. (D) Marker gene expression was compromised in fins1. The gene expression was measured using 5-d-old seedlings grown on MS agar media containing 6% fructose or mannitol. (E) Expression analysis of FINS1 transcripts (RT-PCR and DNA-PCR) and proteins (protein blot) in a selected FINS1-complemented fins1 (cFINS1). (F) cFINS1 seedlings were fructose sensitive similar to WT (7 d). Scale bar, 5mm. (G) Expression analysis of catalytically inactive FINS1_ssm in a selected FINS1_ssm-complemented fins1 (cSSM) seedling. (H) cSSM seedlings were fructose sensitive similar to WT (7d).

Figure 4. FINS1 in fructose signaling is independent of its sucrose metabolic activity.

(A–C) WT, fins1, and cFINS1 seedlings (3.5 d) showed a similar growth phenotype on MS agar plates with glucose or fructose in the dark. Mannitol served as an osmotic control. Scale bar, 5 mm. (D–F) fins1 and gin2 seedlings showed differential responses to 6% (7 d), 10% (3 d), and 12% (3 d) sucrose (Suc) under a 16 h photoperiod. Scale bar, 5 mm.

Figure 5. FINS1 in fructose signaling acts downstream of ABA signaling.

(A) FINS1-overexpressing gin1 (ovFINS1 gin1) seedlings showed fructose sensitivity. Seedlings were grown for 4 d under a 16 h photoperiod. HA-tagged FINS1 expression was shown by protein blot analysis. (B, C) ovFINS1 gin1 seedlings showed hypersensitive responses to subsaturated (0.5 µM) but not to saturated (1 µM) levels of ABA. The seedlings were grown for 9 d under a 16 h photoperiod. (D) All seedlings exhibited normal growth without ABA (5 d). Scale bar, 5 mm.

Figure 6. Function of FINS1 in ABA signaling.

(A) fins1 seedlings showed ABA insensitivity during seedling establishment similar to ctr1. The seedlings were grown for 5 d under a 16 h photoperiod. Scale bar, 5 mm. (B–E) Marker gene expression for ABA signaling (1 µM) was altered in fins1. The experiments were repeated three times with similar results.

Figure 7. Working model of hexose signaling network during A. thaliana early seedling development.

HXK1/GIN2-dependent glucose signaling acts upstream of GIN1/ABA2, while FINS1/FBP functions downstream of GIN1/ABA2. Glucose signaling may also integrate into GIN1/ABA2 directly. Only a minor portion of fructose signaling may occur via GIN2/HXK1. Both hexose signals positively and negatively modulate ABA and ethylene responses, respectively.