The mRNA-binding protein HLN1 enhances drought stress tolerance by stabilizing the GAD2 mRNA in Arabidopsis

Abstract

Drought is a common environmental condition that significantly impairs plant growth. In response to drought, plants close their stomata to minimize transpiration and meanwhile activate many stress-responsive genes to mitigate damage. These stress-related mRNA transcripts require the assistance of RNA-binding proteins throughout their metabolic process, culminating in protein synthesis in the cytoplasm. In this study, we identified HLN1 (Hyaluronan 1), an RNA-binding protein with similarity to the animal hyaluronan-binding protein 4 / serpin mRNA binding protein 1 (HABP4/SERBP1), as crucial for plant drought tolerance. The hln1 loss-of-function mutant exhibited higher transpiration rates due to impaired stomatal closure, making it highly susceptible to drought. Drought stress increased HLN1 expression, and the protein underwent liquid-liquid phase separation (LLPS) to form mRNA-ribonucleoprotein (mRNP) condensates in the cytoplasm under osmotic stress. We identified GAD2 as a potential mRNA target of HLN1. GAD2 encodes the predominant glutamate decarboxylase synthesizing γ-aminobutyric acid (GABA), a non-proteinogenic amino acid that modulates stomatal movement. RIP-qPCR and EMSA showed that HLN1 binds GAD2 mRNA, which promotes HLN1 condensate formation. In hln1 mutants, GAD2 transcripts were less stable, reducing steady-state mRNA levels. As a result, hln1 accumulated less GABA and exhibited impaired stomatal closure under drought. Conversely, HLN1 overexpression stabilized GAD2 mRNA, increased GABA levels, and enhanced drought tolerance in transgenic plants. GAD2 overexpression in hln1 mutants also rescued the drought-sensitive phenotypes. Overall, our study reveals a mechanism whereby HLN1 stabilizes GAD2 mRNA to enhance GABA production and drought tolerance. These findings provide novel strategies for engineering drought-resistant crops.

Keywords: Condensate; Drought; GABA; HLN1; mRNA stability; mRNA-binding protein.

Fig. 1

HLN1 is required for drought stress response and tolerance in Arabidopsis. A Morphology of Col-0, hln1, complementation line #1 and #2 (Comp #1 and #2) plants before (upper panel) and after (middle panel) a 20-day drought stress, and 2 days after rewatering (lower panel). B Stomatal conductance in rosette leaves of the indicated plants following drought treatment. Measurements were taken every two days, and data are means ± standard deviations (SD) from three biological replicates. C Stomatal morphology of the indicated genotypes under drought treatment. Leaves from 3-week-old plants were excised after 10 days of drought treatment and imaged by light microscope. Experiments were repeated three times with similar results and representative images are shown. Scale bar = 10 μm. D Stomatal aperture (expressed as width/length ratio) in leaves of the indicated plants under drought stress. Experiments were repeated three times and stomatal apertures from more than 100 stomata were calculated and shown as dots. Double asterisks (**) represent a p-value < 0.01 by Student’s t-test. E Relative water content (RWC) in rosette leaves of the indicated plants after 10-day drought treatment. Data are means ± SD from three biological replicates. Asterisks (*) indicate a p-value < 0.05 by Student’s t-test. F Hydrogen peroxide (H₂O₂) content in drought-treated plants compared to well-watered controls. Data are means ± SD from three biological replicates. Asterisks (*) indicate a p-value < 0.05 by Student’s t-test. G Malondialdehyde (MDA) content in drought-treated plants compared with well-watered plants. Data are means ± SD from three biological replicates. Double asterisks (**) represent a p-value < 0.01 by Student’s t-test. Plant genotypes: Col-0 (wild type); hln1 (hln1 mutant); Comp #1 and #2, two independent complementation lines (i.e., pHLN1-HLN1/hln1 #1 and #2)

Fig. 2

Expression pattern and subcellular localization of HLN1. A-F pHLN1:GUS expression in 7-day-old seedling (A), cotyledons (B), lateral roots (C), root tip (D), guard cells (E), and trichomes (F). G-J pHLN1:GUS expression in the inflorescence (G), rosette leaf (H), flower (I), and silique (J) of 3-week-old plants. Scale bars: 1000 μm (A-D, G-J); 100 μm (E–F). K Subcellular localization of HLN1 in the root tip cells of pGWB 541-HLN1 and pGWB 542-HLN1 transgenic plants. Scale bar in the lower panel (K) represents 20 μm

Fig. 3

HLN1 forms cytoplasmic condensates via phase-separation both in vivo and in vitro. A Fluorescence Recovery After Photobleaching (FRAP) analysis of a HLN1 condensate (arrowhead) in the root elongation zone of an EYFP-HLN1 transgenic seedling. Scale bar = 20 μm. B FRAP recovery kinetics of EYFP-HLN1 condensates in transgenic seedlings. The half-time recovery (t_1/2) was calculated from averaged fluorescence intensity. Error bars represent standard deviations (SD) from 9 biological replicates. C in vitro phase separation of HIS-EYFP and HIS-EYFP-HLN1 proteins following the addition of PEG 8000. Scale bar = 20 μm. D Concentration-dependent formation of HLN1 condensates with PEG 8000. Scale bar = 20 μm. E FRAP analysis of a representative HIS-EYFP-HLN1 droplet. scale bar = 20 μm. F FRAP recovery plot of HIS-EYFP-HLN1 droplets. Half-time recovery (t₁/₂) was calculated from averaged intensities. Error bars represent SD from 9 biological replicates

Fig. 4

HLN1 interacts with GAD2 mRNA to regulate its stability in Arabidopsis. A Subcellular localization of HLN1 in root tip cells of transgenic lines under normal conditions, mannitol treatment, and mannitol treatment combined with cycloheximide (CHX) treatment. Scale bar = 10 μm. B RIP-qPCR analysis of EYFP-HLN1 binding to GAD2 mRNA with or without dehydration treatment. EYFP-EYFP transgenic line was used as a negative control. Data are means ± SD (n = 3 biological replicates). **, p < 0.01 by Student’s t-test. C EMSA of HLN1 binding to GAD2 3’UTR analyzed by native PAGE. MBP-GST was negative control. D GAD2 mRNA decay kinetics in hln1 mutants. Data points show means ± SD (n = 3). E GAD2 mRNA decay kinetics in HLN1 overexpression (OE) lines. Two independent lines showed similar results. Data from one line shown (means ± SD, n = 3 per time point). F Relative expression of GAD2 mRNA in Col-0, hln1, and two complementation lines (Comp #1 and #2) under drought stress (means ± SD, n = 3). **, p < 0.01 by Student’s t-test. G GABA content in Col-0, hln1 and two complementation lines (Comp #1 and #2) under control or drought stress conditions (mean ± SD, n = 3). **, p < 0.01 by Student’s t-test

Fig. 5

Exogenous GABA treatment reduces stomatal conductance and drought-induced damage in the hln1 mutant. A Stomatal morphology of the indicated genotypes under drought or GABA treatment. Representative results of three independent experiments are shown. Scale bar = 10 μm. B Stomatal apertures (expressed as width/length ratio) of the indicated genotypes with or without drought or GABA treatment. Experiments were repeated three times and stomatal apertures from more than 100 stomata were calculated. C Stomatal conductance in 3-week-old plants of the indicated genotypes under drought or 4 mM GABA treatment (means ± SD, n = 3). D Malondialdehyde (MDA) content in 3-week-old plants of the indicated genotypes under drought or 4 mM GABA treatment (means ± SD, n = 3). E Hydrogen peroxide (H₂O₂) content in 3-week-old plants of the indicated genotypes under drought or 4 mM GABA treatment (means ± SD, n = 3). Genotypes include Col-0 (wild type); hln1(hln1 mutant); Comp #1 and #2 (two independent hln1 complementation lines, i.e., pHLN1-HLN1/hln1 #1 and #2). Error bars represent standard deviation (SD). The double asterisks (**) indicate a p-value < 0.01 and the single asterisk (*) indicates a p-value < 0.05 by Student’s t-test

Fig. 6

Overexpression of GAD2 enhances drought tolerance of the hln1 mutant. A Morphology of 3-week-old plants of the indicated genotypes before (upper panel) and after (middle panel) 20-day drought stress treatment, and 2 days after rewatering (lower panel). B Stomatal conductance in rosette leaves after drought treatment. C Relative water content (RWC) in rosette leaves after 10-day drought treatment. D Stomatal morphology in rosette leaves under drought treatment. Leaves were excised and imaged immediately with a light microscope after 10-day drought treatment. More than 100 stomata of each genotype were measured, and the experiments were repeated three times with similar results. Scale bar = 10 μm. E Stomatal apertures (expressed as width/length ratio) under control or drought treatment. Experiments were repeated three times and stomatal apertures from more than 100 stomata were calculated. F Hydrogen peroxide (H₂O₂) content in rosette leaves under control or drought treatment. G Malondialdehyde (MDA) content in rosette leaves under control or drought treatment. H GABA content in rosette leaves under control or drought treatment. Plant genotypes include Col-0 (wild type); hln1 (hln1 mutant); GAD2/hln1 OE #1 and #2 (two independent hln1 complementation lines). Data in (B-C and F-G) are means and standard deviation (SD) from three biological replicates. The double asterisks (**) indicate a p-value < 0.01, and the single asterisk (*) indicates a p-value < 0.05 by Student’s t-test

Fig. 7

HLN1 condensates stabilize GAD2 mRNA during drought stress. Glutamate decarboxylase 2 (GAD2) catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA) in leaves. Under well-watered conditions, GAD2 transcript levels remain low, supporting basal GABA synthesis. During drought stress, GAD2 expression increases. HLN1 binds with GAD2 mRNA and other transcripts and undergoes liquid–liquid phase separation (LLPS) to form the mRNA-protein (mRNP) condensates that protect GAD2 mRNA from rapid degradation. This stabilization enables sustained and elevated GAD2 protein production, enhancing GABA synthesis under drought conditions. In the absence of HLN1, GAD2 mRNA becomes less stable, reducing GABA levels and impairing stomatal closure during drought stress