High temperature is one of the major environmental stresses affecting plant growth and fitness. Heat stress transcription factors (HSFs) play critical roles in regulating the expression of heat-responsive genes. However, how HSFs are regulated remains obscure. Here, we show that ALBA4, ALBA5 and ALBA6, which phase separate into stress granules (SGs) and processing bodies (PBs) under heat stress, directly bind selected messenger RNAs, including HSF mRNAs, and recruit them into SGs and PBs to protect them from degradation under heat stress in Arabidopsis. The alba456 triple mutants, but not single and double mutants, display pleiotropic developmental defects and hypersensitivity to heat stress. Mutations in XRN4, a cytoplasmic 5′ to 3′ exoribonuclease, can rescue the observed developmental and heat-sensitive phenotypes of alba456 seedlings. Our study reveals a new layer of regulation for HSFs whereby HSF mRNAs are stabilized by redundant action of ALBA proteins in SGs and PBs for plant thermotolerance.
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The eCLIP-seq sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under the accession number PRJNA750641. The eCLIP-seq data for Pro35S:GFP at 22 °C were downloaded from GEO (GSE128619). No new code was generated in this study. The accession numbers of the genes used in this research are as follows: ALBA4 (AT1G20220), ALBA5 (AT1G76010), ALBA6 (AT3G07030), DCP5 (AT1G26110), PAB2 (AT4G34110), PAB4 (AT2G23350), Rbp47b (AT3G19130), DCP1 (AT1G08370), DCP2 (AT5G13570), EIF4A1 (AT3G13920), PAT1 (AT1G79090), XRN4/EIN5 (AT1G54490), HSP101 (AT1G74310), HSFA7B (AT3G63350), HSFA2 (AT2G26150), HSFB2A (AT5G62020), MBF1C (AT3G24500), DREB2A (AT5G05410), ACTIN2 (AT3G18780). Source data are provided with this paper.
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We thank Q.-J. Chen for the CRISPR–Cas9 system. This study was supported by the National Key R&D Programme of China to W.Q. (grant no. 2018YFE0204700) and the National Natural Science Foundation of China to L.-M.F. (grant no. 32170285) and W.Q. (grant no. 31970614).
The authors declare no competing interests.
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a, Yeast two-hybrid analysis of the interactions between ALBA proteins and PB and SG components. Combinations of constructs were transformed into yeast strain AH109 and assayed on medium lacking Leu and Trp (SD-TL), medium lacking Leu, Trp, and His (SD-TLH), and medium lacking Leu, Trp, His and Ade (SD-TLHA). AD, activating domain; BD, binding domain; Vec, empty vector. Data are representative of three independent experiments. b, Co-IP results showing the interactions between ALBA proteins and DCP5 in vivo. Transgenic plants expressing ALBA4-Myc or ALBA5-Myc and F1 offspring seedlings (ProALBA4:ALBA4-Myc × ProDCP5:DCP5-GFP and Pro35S:ALBA5-Myc × ProDCP5:DCP5-GFP) were used for Co-IP. Data are representative of two independent experiments with similar results.
Extended Data Fig. 2 ALBA4, ALBA5 and ALBA6 reversibly localize to cytoplasmic mRNP granules under heat stress.
a, b, Fluorescence images showing colocalization of ALBA6 with ALBA4 (a) and ALBA5 (b). The root tissue of 5-day-old F1 seedlings co-expressing the indicated transgenes under control (22 °C) and heat stress (39 °C for 50 min) conditions was used for the experiments. Scale bars = 10 μm. Images are representative of three independent replicates, and at least five roots were monitored for each condition and replicate. c, Fluorescence images showing localization of ALBA4, ALBA5, ALBA6 and Rbp47b during recovery from heat stress. The transgenic plants were pretreated at 39 °C for 50 min, after which images of root tip cells were taken at 22 °C at the indicated time points. Scale bars = 10 μm. d, Quantification of ALBA4, ALBA5, ALBA6 and Rbp47b foci in (c). Data represent the mean ± SD. For ALBA4-GFP foci, n = 17, 11, 14, 14 and 10, for 10, 30, 50, 70 and 90 min, respectively; for ALBA5-GFP foci, n = 13, 18, 12, 17 and 8, for 10, 30, 50, 70 and 90 min, respectively; for ALBA6-GFP foci, n = 18, 23, 29, 26 and 24, for 10, 30, 50, 70 and 90 min, respectively; for Rbp47b, n = 30, 23, 25, 18 and 15, for 10, 30, 50, 70 and 90 min, respectively.
a, b, Fluorescence images showing colocalization of ALBA5 with SG marker proteins Rbp47b (a) and PAB2 (b). c-e, Fluorescence images showing colocalization of ALBA5 with PB marker proteins DCP1 (c), DCP2 (d) and DCP5 (e). The root tissue of 5-day-old F1 seedlings co-expressing the indicated transgenes under control (22 °C) and heat stress (39 °C for 50 min) conditions was used for the experiments. Scale bars = 10 μm. Images are representative of three independent replicates, and at least five roots were monitored for each condition and replicate.
a, FRAP of ALBA5-GFP foci in the cytoplasm of Arabidopsis root tip cells. Time 0 indicates the time of the photobleaching pulse. Arrowheads indicate the bleached foci. Scale bars = 0.5 μm. Data are representative of four independent experiments with similar results. b, Dynamics of ALBA5-GFP foci in the cytoplasm of Arabidopsis root tip cells after heat stress treatment. Plants were pre-treated at 39 °C for 20 min, and high-resolution images were acquired at 39 °C continuously. Scale bars = 0.5 μm. Data are representative of three independent experiments with similar results. c, FRAP of ALBA6-GFP foci in the cytoplasm of Arabidopsis root tip cells. Scale bars = 0.5 μm. Data are representative of four independent experiments with similar results. d,e, Recovery curves of ALBA5-GFP (d) and ALBA6-GFP (e) cytoplasmic foci after photobleaching. Data are presented as mean ± SD (n = 4). f, Analysis of ALBA4 (16 μM) droplet formation in vitro. Scale bar = 10 μm. Data are representative of three independent experiments with similar results.
a, b, Phenotypes (a) and quantification of the fresh weight (b) of 10-day-old wild-type Col-0, single mutant (alba4, alba5 and alba6), double mutant (alba45, alba46 and alba56) and triple mutant (alba456-3) seedlings. c, d, Primary root growth phenotype (c) and quantification of the primary root length (d) of 8-day-old Col-0 and alba mutant seedlings. In (b) and (d), dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum. Significant differences between two genotypes are indicated by different letters (P < 0.05, one-way ANOVA with post hoc Tukey HSD tests; n = 30). See Source Data for P values.
Extended Data Fig. 6 ALBA4, ALBA5 or ALBA6 transgene can rescue the developmental phenotypes of alba456.
a, b, Representative photographs (a) and quantification of the fresh weight (b) of 10-day-old Col-0, alba456-2 and ALBA4/5/6 complementation lines. GFP-tagged ALBA4 and ALBA5 were driven by their native promoters, and GFP-tagged ALBA6 was driven by the 35 S promoter. c,d, Primary root growth phenotype (c) and quantification of the primary root length (d) of 8-day-old Col-0, alba456-2 and ALBA4/5/6 complementation lines. In (b) and (d), dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum. Significant differences between two genotypes are indicated by different letters (P < 0.05, one-way ANOVA with post hoc Tukey HSD tests; n = 30). See Source Data for P values.
Extended Data Fig. 7 ALBA4-GFP and DCP5-GFP transgenes can rescue the heat hypersensitive phenotype of alba456-2 and dcp5-1, respectively.
a, b, Photographs of Col-0, alba456-2, ALBA4-GFP-complemented alba456-2, hsp101, dcp5-1, DCP5-GFP-complemented dcp5-1 seedlings under control (22 °C), heat shock (44 °C for 90 min) and prolonged heat treatment (37 °C for 5 d). Plants in each treatment group were grown on the same plate. At least three independent biological replicates were conducted. Results from one representative biological replicate are shown. The hsp101 mutant was included as a control. Scale bars = 2 cm. c, Quantification of the data shown in (a) and (b). The percentage of seedlings in different phenotypic classes was determined for different genotypes and different conditions. Data are presented as the mean ± SD of three biological replicates. Significant differences between two groups are marked with different letters as determined by two-sided Fisher’s exact test (P < 0.01). See Source Data for P values.
a, Coomassie blue staining of His-tagged ALBA5 purified from E.coli. Data are representative of five independent experiments with similar results. b, EMSA results showing ALBA5 binding to ssRNA probes and competition by unlabeled ssRNA probes. 3′-Fam-labeled substrates (80 nM) were incubated with increasing concentrations of ALBA5. The protein concentrations in lanes 1, 2, 3 and 4 were 0, 1, 2 and 3 μM, respectively. For competition analysis, 32 μM of unlabeled substrate was used as a cold probe and incubated with 3 μM of ALBA5-His and 80 nM of 3′-Fam-labeled substrates. For EMSA, at least three independent biological replicates were performed, and representative results are shown. c, Principal component analysis (PCA) plots of ALBA5 binding clusters in 5-day-old seedlings under control (22 °C) and heat stress (39 °C for 50 min) conditions.
a, Schematic presentation of the Arabidopsis XRN4 gene. Untranslated and coding regions are depicted as thick gray and blue boxes, respectively. Introns are depicted as thin black boxes. Triangles indicate the CRISPR/Cas9 target sites and T-DNA insertion position in XRN4. The XRN4 gene is drawn to scale. The sgRNA target sequences are highlighted in yellow. The PAM sites are underlined. b, Photographs of Col-0, alba456-2, xrn4-5 and alba456-2 xrn4-9 seedlings under control (22 °C) and prolonged heat treatment (37 °C for 5 d). Scale bars = 2 cm. c, Quantification of the data shown in (b); the percentage of seedlings in different phenotypic classes was determined for different genotypes and different conditions. Data are presented as the mean ± SD of three biological replicates. Significant differences between two groups are marked with different letters as determined by two-sided Fisher’s exact test (P < 0.01). d, Decay profiles of HSF mRNAs under heat stress conditions (37 °C for 12 h). ALBA5 non-target AT2G07689 was used as the negative control. The mRNA decay profile of each genotype is presented as the relative mRNA abundance after transcription inhibition by cordycepin. Results from one representative replicate of two independent biological replicates are shown (each in three technical replicates). Data represent mean ± SD. See Source Data for P values.
Supplementary Figs. 1–8.
Identification of co-purified proteins of ALBA4, ALBA5, ALBA6, DCP5 and Rb47b by mass spectrometry.
List of ALBA5 binding sites from eCLIP-seq.
Primers, sgRNAs and probes used in this study.
Statistical source data for Supplementary Figures.
Statistical source data for Fig.3.
Statistical source data for Fig.4.
Uncropped scans of gels for Fig. 5g.
Statistical source data for Fig.6.
Statistical source data for Fig.7.
Uncropped western blots for Extended Data Fig. 1b.
Statistical source data for Extended Data Fig.5.
Statistical source data for Extended Data Fig.6.
Statistical source data for Extended Data Fig.7.
Statistical source data for Extended Data Fig.9.
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Tong, J., Ren, Z., Sun, L. et al. ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules. Nat. Plants 8, 778–791 (2022). https://doi.org/10.1038/s41477-022-01175-1