PAT mRNA decapping factors are required for proper development in Arabidopsis

Evolutionarily conserved protein associated with topoisomerase II (PAT1) proteins activate mRNA decay through binding mRNA and recruiting decapping factors to optimize posttranscriptional reprogramming. Here, we generated multiple mutants of pat1, pat1 homolog 1 (path1), and pat1 homolog 2 (path2) and discovered that pat triple mutants exhibit extremely stunted growth and all mutants with pat1 exhibit leaf serration while mutants with pat1 and path1 display short petioles. All three PATs can be found localized to processing bodies and all PATs can target ASYMMETRIC LEAVES 2‐LIKE 9 transcripts for decay to finely regulate apical hook and lateral root development. In conclusion, PATs exhibit both specific and redundant functions during different plant growth stages and our observations underpin the selective regulation of the mRNA decay machinery for proper development.

As mentioned above, Arabidopsis encodes three PAT1 paralogs.PAT1 specifically functions in response to osmotic stress [14] while all three PAT proteins function redundantly during Turnip mosaic virus infection [22].However, more specific functions of these three PATs during plant development have not been studied in detail.Here, we mutated all three PAT genes in the summ2 background [11] to avoid autoimmunity interference.We found that all three PAT proteins interacted with the LSM1 decapping factor and localized to PBs upon different stimuli perception.Based on our examination of the developmental phenotype of multi-pat mutants combined with RNA-seq to examine differently expressed genes, we conclude PAT1 is mainly responsible for keeping regular leaf patterns and working redundantly with the other two PATs during plant development.

Plant treatment and confocal microscopy
Seedlings were grown in liquid MS medium for 5 days, confocal microscopy pictures were taken with a Zeiss LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) after 20 min treatment with MS containing 0.5 lM IAA, 1 lM cytokinin, 25 lM ACC, 5 lM flg22, or 0.1 mM ATP.
Ethylene triple-response assays were conducted as described [15].Apical hook (AH) angles were defined as 180°minus the angle between the tangential of the apical part with the axis of the lower part of the hypocotyl; 180°p lus that angle is defined as the angle of exaggerated hook curvature [24].Cotyledon and hook regions of etiolated seedlings were collected for XRN1 assays.

RNA-seq analysis
Total RNA was extracted from 6-week-old soil-grown plants including summ2-8, pat1-1summ2-8, path1-4summ2-8, path2-1 summ2-8, pat1-1path1-4summ2-8, pat1-1path2-1summ2-8, path1-4path2-1summ2-8, and pat1-1path1-4path2-1summ2-8 using the NucleoSpinÒ RNA Plant kit (Machery-Nagel, D€ u ren, Germany).RNA quality control, library preparation, and sequencing were performed by BGI.The reads obtained from the instrument were base called using the instrument manufacturer's base calling software, then the reads were mapped to the A. thaliana TAIR10 reference genome with STAR (version 2.5.1b) using two-pass alignment mode [25].The read counts for each gene were detected using HTSEQ (version 0.5.4p3) and summarized in Table S3 [26].The Araport11 annotation was used for both mapping and read counting.The counts were normalized using the TMM normalization from EDGER package in R [27].PCA analysis was performed using DESEQ2 package in R [28].Prior to statistical testing, the data were voom transformed and then the differential expression between the sample groups was calculated with LIMMA package in R [29,30].Genes with fold change ≥ 2 or ≤ À2 and P-value ≤ 0.05 are listed in Data S1.Functional Annotation Tool DAVID Bioinformatics Resources 6.8 has been used for GO term analysis for the differently expressed genes using detected genes as background [31].The high-throughput RNA-seq data have been deposited in NCBI SRA database with accession number PRJNA1006171 and processed data to GEO with accession number GSE253757.

RNA extraction and qRT-PCR
Total RNA from tissues was extracted with TRIzol TM Reagent (Thermo Fisher Scientific), 2 lg total RNA was treated with DNAse I (Thermo Fisher Scientific), and reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer's instructions (Thermo Fisher Scientific).The ACT2 gene was used as an internal control.qPCR analysis was performed on a Bio-RAD (Hercules, CA, USA) CFX96 system with SYBR Green Master Mix (Thermo Scientific).Primers are listed in Table S2.

In vitro XRN1 susceptibility assay
Transcripts XRN1 susceptibility was determined as described [15].Total RNA was extracted using the NucleoSpinÒ RNA Plant kit (Machery-Nagel).One microgram RNA was incubated for 2 h at 37 °C with either 1 unit of XRN1 (New England Biolabs, Ipswich, MA, USA) or water; after heating inactivation under 70 °C for 10 min, the digest was then reverse transcribed into random primed cDNA.Capped target transcript accumulation was measured by comparing transcript levels from XRN1-treated versus water-treated samples using qRT-PCR for the individual samples, non-decapping target EIF4A1 serves as inner control [15].

RIP assay
RIP was performed as previously described [15].Transcript levels in input and IP samples were quantified by qRT-PCR, and levels in IP samples were corrected with their own input values and then normalized to MYC-YFP lines for enrichment.

Statistical analysis
Statistical details of assays are reported in the figures and legends.SYSTAT software (Systat Software Inc., San Jose, CA, USA) was used for data analysis.Statistical significance was determined by one-way ANOVA (analysis of variance) followed by Holm-Sidak test.

PAT mRNA decapping factors interact and co-localize with LSM1
The Arabidopsis genome encodes three PAT mRNA decapping factors: PAT1 (AT1g79090), PATH1 (AT3g22270), and PATH2 (AT4g14990) [11].We previously reported that PAT1 localizes to PBs and can be found in complexes together with the decapping component LSM1 [11].To test if PATH1 and PATH2 are also found in mRNA decay complexes, we transiently expressed all three PAT-HA fusions with YFP-LSM1 and performed co-immunoprecipitation assays.All three PAT-HA fusions could be detected in LSM1 immunoprecipitates (Fig. 1A), indicating that, similar to PAT1, PATH1 and PATH2 also localize to LSM1-positive compartments.To confirm this, we also performed bimolecular fluorescence complementation (BiFC) analysis.As expected, cCFP-LSM1 and all three nCFP-PATs but not nCFP-GUS were able to reconstitute CFP in distinct foci localized in the cytoplasm, most likely PBs.Furthermore, upon auxin treatment, the number of CFP foci reconstituted by LSM1 and all three PATs were increased (Fig. 1B).In line with this, a previous mass spectrometry study detected all three PAT proteins in LSM1 precipitates [36].

PATs localize in cytoplasmic foci PBs
mRNA decapping components and mRNAs can concentrate into PBs in the cytoplasm [3].Our BiFC data indicate transiently expressed PATs can concentrate to PBs together with LSM1.To investigate if all three PAT proteins may also form PBs in Arabidopsis, we exposed seedlings expressing either Venus fusions of the PAT proteins (Fig. S1) or GFP fusions with the LSM1 protein [12] to the phytohormones auxin (IAA), cytokinin, ethylene precursor ACC, bacterial flagellin peptide flg22, and extracellular ATP in liquid MS medium.All Venus-PATs proteins and LSM1-GFP form very few PBs under non-treatment (NT) condition; however, all stimuli induced massive increase in Venus-PAT1 and LSM1-GFP distinct foci, resembling PBs in roots within 20 min (Fig. 2) while PATH1 and PATH2 also formed PB-like structures in response to IAA, flg22, and ACC treatment but to a lesser extend (Fig. 2).To test if auxin could enhance the PATs-LSM1 interaction, we conducted co-IP assays on IAA-treated samples expressing PATs-HA and YFP-LSM1 (Fig. S2).The ratio of PAT1-HA/YFP-LSM1 and PATH1-HA/ YFP-LSM1 in GFP-IP samples did not change after IAA treatment, but PATH2-HA/YFP-LSM1 slightly increased.Nevertheless, these observations indicate PAT1 as the main PB component compared to PATH1 and PATH2.

Characterization of multiple PAT(s) loss-of-function mutants
While we had detected PAT1 in PBs and found it under SUMM2 surveillance [11], little was known about mRNA targets or functions of the three PATs in Arabidopsis.To address this, we generated single, double, and triple knockouts (KOs) of the three PATs in the summ2-8 background to avoid immune activation.path1 and path2 mutants were therefore generated in pat1-1summ2-8 and summ2-8 by CRISPR/CAS9mediated genome editing (Fig. 3A, Fig. S1A).
Figure S1B shows the mutations of PATH1 and PATH2 in two independent pat triple mutants.The growth phenotypes of 6-week-old soil-grown plants of pat single, double, and triple mutants at 21 °C with 8/16 h light/ dark photoperiod are shown in Fig. 3A and Fig. S1A.Both independent lines of pat1-1path1-4path2-1summ2-8 and pat1-1path1-5path2-2summ2-8 exhibited markedly stunted growth compared to the other pat single or double mutants (Fig. 1C, Fig. S1A,B).Interestingly, both PAT1 and PATH1 are expressed at higher transcriptional levels when the other two PATs are non-functional (Fig. S1C).All these data indicate functional redundancy of PATs in regulating plant development.

All three PATs redundantly regulate plant development
To better quantify growth phenotype of pat mutants, we dissected 6-week-old plants of Col-0 and multiple pat mutants (Fig. 3B) and recorded plant leaf numbers in different developmental stages from 1-to 6-week-old (Fig. 3C).All mutants except path1-4summ2-8 and path2-1summ2-8 had developed significantly fewer leaves than Col-0 and summ2-8 from 4 weeks old, and the leaf number difference became more pronounced along with the developmental stages.Among all the pat mutants, pat1-1path1-4path2-1summ2-8 exhibited the lowest   amount of leaf numbers (Fig. 3C), indicating some functional redundancy of all three PATs in leaf emergence.

PATs play different roles in regulating leaf morphology
Leaf morphology is an essential part of phytomorphology.We therefore also inspected leaf size and shape of the latest expanded leaves from the 6-week-old plants of multiple pat mutants.Similar to leaf number, pat1-1path1-4path2-1summ2-8 exhibited the shortest leaf blade and all mutants except path1-4summ2-8 and path2-1summ2-8 developed shorter leaf blade than Col-0 and summ2-8 (Fig. 3D).Petioles connect leaf lamina to the plant stem, pat1-1summ2-8 had shorter petioles than Col-0 and summ2-8, while pat1-1path1-4summ2-8 and pat1-1path1-4path2-1summ2-8 had the shortest petioles (Fig. 3E).Overall, pat1-1path1-4summ2-8 exhibit lowest petiol-blade ratio (PBR) and pat1-1path2-1summ2-8 the highest PBR (Fig. 3F), indicating three PATs function redundantly in regulating leaf blade size and only PAT1 and PATH1 are involved in the development of leaf petiole length in which PAT1 most likely plays the main role.Evolutionarily, variation in plant leaf shape reflects natural selection operating on function.To better understand PATs function in leaf serration, we counted serration numbers per half leaf and measured serration level on the latest expanded leaves from the multiple pat mutants [37].Interestingly, pat1-1summ2-8 displayed increased number of serrations compared to control plant while pat1-1path1-4summ2-8 but not pat1-1path2-1summ2-8 had most serrations (Fig. 3G).All mutant combinations with pat1 loss of function exhibited higher serration levels than any other mutant combination and pat1-1path2-1summ2-8 displayed lowest serration level in plants with PAT1 mutated (Fig. 3H), indicating PAT1 is regulating leaf serration level, and PATH2 may play opposite role.Furthermore, complementation lines of pat1-1path1-4summ2-8 and pat1-1path2-1summ2-8 with Venus-PATH1 and Venus-PATH2, respectively, exhibit the same growth phenotype as pat1-1summ2-8 (Fig. S1D,E).All these results suggest that the functions of PATH1 and PATH2 are partially redundant to the other PATs, while PAT1 serves a main and specific function in development.

PAT(s) loss-of-function mutants exhibit different transcriptomic profiles
To identify genes which affect different developmental programs regulated by PATs, we performed RNA-seq from plants of all pat single, double, and triple mutant combinations shown in Fig. 3A.Principal component analysis (PCA) plot (Fig. 4A) exhibits general similarities of these eight lines, revealing that mutants with pat1 are separated from the other plants based on PC1, and based on PC2, pat double mutants with path2 are more similar to each other compared to any other mutants.Data S1 shows differently expressed genes in multiple pat mutants which are clustered in Fig. 4B, and numbers of shared differently expressed genes (DEGs) are summed up in Fig. 4C.pat1 single and double mutants have more DEGs than path1 and path2 single and double mutants while pat triple mutants accumulate most misregulated genes.Among these DEGs in pat triple mutants, 208 genes (116 upregulated and 92 downregulated genes) are specifically related to pat1 loss of function, and 457 genes are associated with mutations of both PAT1 and PATH1 while 356 genes are misregulated only when all three PATs are non-functional (Fig. 4C).GO term analysis on misregulated genes in pat1-1path1-4path2-1summ2-8 showed that: (a) genes involved in oxidation reduction are largely misregulated, (b) transcripts involved in oxidative stress response accumulated, and (c) transcripts responsible for auxin response and signaling and growth regulation are reduced in pat triple mutant (Fig. S3) [31].

PATs regulate different genes during leaf development
Leaf initiation and outgrowth have been reported under auxin regulation [38].We therefore took a closer look at the auxin responsive genes including SAURs (SMALL AUXIN UP RNAs), IAAs, MYBs (MYB DOMAIN PROTEINs), WAG1, WAG2, etc. and performed qRT-PCR to confirm their expression levels (Fig. 4D,E).They were least expressed in pat triple mutant compared to all pat double mutants and pat1 single mutant in which the auxin responsive genes were also down-regulated compared to summ2-8 and path1 and path2 single mutants (Fig. 4D,E).Since we could already observe leaf number difference in 2-week-old pat mutants (Fig. 3C), we checked the expression of auxin responsive genes at an early developmental stage where there were only cotyledons developed.Interestingly, the auxin responsive genes SAUR23, IAA19, and IAA29 are downregulated in 1-week-old plants of the pat1 single, double, and triple mutants (Fig. S4A), indicating decreased auxin signaling might be responsible for the reduced leaf number phenotype in pat mutants.
The NAC family member CUP-SHAPED COTY-LEDON 2 (CUC2) and PIN1 auxin efflux carrier have been modeled to explain leaf serration and CUC3 has also been reported to be essential to keep serration [41,42]; however, both RNA-seq and our qRT-PCR results indicated that CUC2 and CUC3 were downregulated in the serrated mutant pat1-1summ2-8 while we found other two NAC family genes ANAC003 and ANAC016 specifically accumulated in all serrated mutants indicating PAT1 and PATH1 might regulate other NAC genes expression during leaf patterning (Fig. 4D,E).Nevertheless, all these results indicate all three PATs regulate different genes during leaf development.needed for plant development.Besides leaf development, we have reported pat triple mutant exhibits deficiencies in making AH and lateral roots (LRs) partially due to misregulation of the mRNA decapping target gene ASL9.To assess the function of each PAT mRNA decay factor during apical hooking, we germinated seedlings of pat single, double, and triple mutants in darkness in the presence or absence of ethylene precursor ACC [15].Upon ACC treatment, all the pat double mutants, especially pat1-1path2-1summ2-8, form less stringent AHs than any other pat single mutants and the summ2-8 control plants, while pat triple mutants hold the strongest hookless phenotype (Fig. 5A,B).Furthermore, pat1 double mutants had reduced amount of LRs compared to the control and pat single mutants while LRs were almost absent in pat triple mutants (Fig. 5C,D).Collectively, these data indicate all three PATs, and especially, PAT1 and PATH2 are essential for AH and LR development.

All three
To test if ASL9 is misregulated in pat single and double mutants, we performed qRT-PCR in ACC-treated pat mutants.Interestingly, compared to summ2-8, ASL9 accumulated in pat1-1path2-1summ2-8 mutant, although the expression levels were not as high as the ones in pat triple mutants under both mock and ACC treatment conditions (Fig. 5E).To determine if ASL9 is a common target of all three PATs factors, we assayed for capped ASL9 transcripts in ACC and mock-treated pat mutants [15].We verified that with ACC treatment, pat triple mutants accumulated significantly higher levels of capped ASL9 transcripts than all lines but pat1-1path2-1summ2-8 (Fig. 5F), although only the former was statistically different from the summ2-8 control.Moreover, we also performed RNA immunoprecipitation (RIP) and verified in all Venus-PAT lines compared to a free YFP control line (MYC-YFP) (Fig. 5G), the enrichment fold numbers indicated stronger binding efficiency of ASL9 for PAT1 and PATH2 than for PATH1.These data confirm that ASL9 mRNA can be found in all three PAT complexes, and that mRNA decapping regulates ASL9 mRNA levels during AH and LR formation, preferentially but not exclusively by PAT1 and PATH2.

Discussion
Plant development requires massive overhauls of gene expression [43].Loss of function of all three PATs causes most severe dwarfism and deficiencies in AH and LR development compared to any single or double mutants; and pat triple mutant exhibits the most differently regulated genes of which we found 356 genes specifically misregulated in pat triple mutants (Fig. 4C).In addition, pat triple mutants expressed the lowest level of auxin-responsive and shade avoidance-related genes in 6-week-old plants (Fig. 4D,  E) but accumulated the highest level of ASL9 during AH formation (Fig. 5E), indicating some redundancy and sub-functionalization among the three PATs in leaf, AH, and LR development [44].The observation that all three PATs localized into PBs in response to IAA treatment could also indicate functional redundancy of the PAT proteins (Figs 1B and 2).Our phenotypic characterizations also showed that compared to pat1-1summ2-8, leaf serration and dwarfism are more severe in pat1-1path1-4summ2-8, while pat1-1path2-1summ2-8 exhibits less serration and normal petioles, indicating that PAT1 and PATH1 share some functions in leaf serration and petiole elongation while PATH2 may have opposite functions (Fig. 3).Additionally, our RNA-seq analysis showed path2 double and triple mutants were separated from other plants based on PC2 in PCA plot (Fig. 4A) and shared DEGs analysis manifests more DEGs shared by pat1-1path1-4summ2-8 and pat triple mutant than the other two pat double and triple mutants.Moreover, 28 genes were misregulated only in pat1 and pat1path1 double mutants but not when path2 was mutated in these backgrounds (Fig. 4C).Collectively, these data indicate that mutations in PAT1 and PATH1 provide the strongest contribution to the growth phenotypes of pat triple mutant and PATH2 may have other specific/opposite functions.However, pat1-1path2-1summ2-8 exhibits less stringent AH, fewer LR, and higher level of ASL9 expression compared to any other pat single and double mutants.Together with the fact that PAT1 and PATH2 bind more ASL9 transcripts, these data suggest PAT1 and PATH2 exhibit a main function in ASL9 decapping and decay during AH and LR development processes.
Interestingly, pat1 single mutants still exhibit dwarfism and leaf serration, while neither path1 nor path2 single mutants exhibit developmental defects (Fig. 3).Our RNA-seq data show that all mutants with pat1 loss of function are separated from other mutants based on PC1 in PCA plot; 482 transcripts are differentially expressed in pat1 single mutants compared to only 54 genes in path1 and 279 in path2 single mutants (Fig. 4C).While we do not know how these differences contribute to the developmental phenotype of pat1, these observations suggest a specific and principal function of PAT1 during plant development.In line with this, we previously reported that PAT1 specifically functions in response to osmotic stress [14] while all three PAT proteins function redundantly during Turnip mosaic virus infection [22].
Besides pat triple mutants, other mRNA decapping deficient mutants also exhibit abnormal development or postembryonic lethality [13,15,45,46].The stunted growth phenotype and downregulation of developmental and auxin-responsive mRNAs in the pat triple mutant (Figs 3 and 4) may support a model in which defective clearance of suppressors of development hampers developmental changes.
Conserved PAT proteins bind LSM1-7 complex through LSM2/LSM3 and couple mRNA deadenylation and decapping [47].However, we found all three Arabidopsis PATs can be found in complexes with LSM1.Furthermore, auxin treatment did not affect the ratio of PAT1/LSM1 or PATH1/LSM1 complexes, indicating PATs and LSM1-7 are typically integrated (Fig. S2).However, the ratio between PATH2 and LSM1 was slightly increased by auxin treatment (Fig. S2), suggesting PATH2, as an mRNA decapping component, might have additional functions.Among all three PATs, PAT1 responds to broad stimuli and re-localizes to PBs while PATH2 exhibits response to ACC in limited cells, indicating a specific role of PATH2 in ethylene response (Fig. 2).The release of mRNA from polysomes or so-called ribosome-free mRNPs induces PB formation [1], indicating PAT1 could bind broader mRNAs compared to PATH1 and PATH2 and form PBs more efficiently.Moreover, yeast PAT1 enhances the condensation of DHH1, and RNA and PAT1-DHH1 interaction is essential for PB assembly [48].Arabidopsis PAT1 might interact more competently with DHH1 compared to PATH1 and PATH2 and promote PB formation more efficiently.Several studies also implicated yeast PAT1-LSM1-7 complexes in inhibiting exosome-mediated 3 0 -5 0 decay leading to stabilization and accumulation of certain mRNAs [49,50].Therefore, Arabidopsis pats loss-of-function mutants may also lose protection of some transcripts from exosome-mediated decay and these destabilized transcripts could be auxin-responsive and growth-related genes.
In conclusion, we have shown that PAT1, PATH1, and PATH2 interact with the LSM1 decapping factor and localize to PBs upon different stimuli perception (Figs 1A,B and 2).Through observing developmental phenotype of multi-pat mutants, investigating cellular localization of PAT proteins, performing RNA-seq to examine differently expressed genes, and conducting XRN1 and RIP assays to study decapping activity of PATs, we identified PAT1 serves a main role during development and functions redundantly with the other two PATs, primarily PATH1 in leaf morphology and PATH2 in targeting ASL9 for decapping and decay during AH and LR development.

1009FEBS
Letters 598 (2024) 1008-1021 ª 2024 The Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

1010FEBS
Letters 598 (2024) 1008-1021 ª 2024 The Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Fig. 1 .
Fig. 1.PAT mRNA decapping factors interact and co-localize with LSM1.(A) Co-IP between the three PAT-HA and YFP-LSM1 fusions.Proteins were transiently co-expressed in Nicotiana benthamiana and tissue was harvested 3 days postinfiltration.Immunoblots of inputs (left panels) and GFP IPs (right panels) were probed with anti-HA antibodies (upper panel) and anti-GFP antibodies (middle panel for YFP-GUS and bottom panel for YFP-LSM1).(B).Bimolecular fluorescence complementation assay shows co-localization of PATs and LSM1, especially after IAA treatment.Proteins were transiently co-expressed in N. benthamiana, and 2 dpi leaves were infiltrated with 0.2 lM IAA or mock, and images were taken 20 min after the treatments.Scale bars indicate 10 lm.The assays were repeated three times and representative pictures are shown.

Fig. 2 .
Fig. 2. PATs localize to distinct foci upon stimuli perception.Venus-PATs expressing seedlings in MS growth medium (non-treatment, NT) or 20 min after treatment with MS containing 0.5 lM IAA, 1 lM cytokinin, 25 lM ACC, 5 lM flg22, or 0.1 mM ATP. Treatments were repeated three times, and representative images (A) and quantification of foci (B) are shown.Scale bars indicate 10 lm.All values represent means AE standard deviation (STDEV), sample size (n) = 10.Bars marked with three asterisks (***) are statistically extremely significant (P-value < 0.001), two asterisks (**) are highly significant and one asterisk (*) is significant compared to the non-treatment controls.
PATs target ASL9 for decay during AH and LR formation Our RNA-seq analysis and pat mutants' growth phenotype indicate all three PATs mRNA decay factors are 1015 FEBS Letters 598 (2024) 1008-1021 ª 2024 The Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Fig. 4 .
Fig. 4. RNA-seq analysis on pat mutants.(A) PCA plot for general similarity of pats single, double, and triple mutants with biotriplicates.(B) Heatmap clustering of DEGs which were differently expressed in at least one of the pat mutants, for comparison of pat mutants and summ2-8.Pearson's metrics were used in hierarchical clustering of the genes.In the plot, red indicates high expression and blue low expression.(C) Numbers of shared DEGs in pat mutants.(D) Heatmap clustering of differentially expressed genes including auxin-responsive genes, shade avoidance-related genes, and leaf serration-related genes.In the plot, red indicates high expression and blue low expression.(E) Expression level of WAG1, WAG2, IAA19, IAA29, SAUR23, CUC2, CUC3, ANAC003, ANAC016, PIL2, and XTR7 in pats mutants.All values represent means AE SE from biotriplicate; bars marked with the same letter are not significantly different from each other (P-value > 0.05).

Fig. 5 .
Fig. 5. PATs target ASL9 for decay during AH and LR formation.Hook phenotypes (A) and apical hook angles (B) in triple responses to ACC treatment of etiolated seedlings of Col-0, summ2, and pat single, double, and triple mutants.Experiments were repeated three times, and representative pictures are shown.The scale bar indicates 1 mm.All values represent means AE STDEV (ST), sample size (n) = 10.LR phenotypes (C) and emerged LR density (D) of 10-day-old seedlings of Col-0, summ2, and pat single, double, and triple mutants.Experiments were repeated three times and representative pictures are shown.The scale bar indicates 1 cm.All values represent means AE ST, sample size (n) = 10.ASL9 expression levels (E) and capped ASL9 transcript levels (F) in cotyledons and hook regions of dark-grown pat single, double, and triple mutants under control or ACC treatment.Error bars indicate SE from biotriplicate.(G) All three PATs can bind ASL9 transcripts.Four-day dark-grown plate seedlings of pat1-1/Venus-PAT1, pat1-1path1-4summ2-8Venus-PATH1, and pat1-1path2-1summ2-8/Venus-PATH2 were taken for the RIP assay.ASL9 transcript levels were normalized to those in RIP of MYC-YFP as a non-binding control.ACT2 was used as a negative control.Error bars indicate SE from biotriplicate, bars marked with the same letter are not significantly different from each other (P-value > 0.05), and bars marked with three asterisks (***) are statistically extremely significant (P-value < 0.001) compared to the MYC-YFP controls.

1018FEBS
Letters 598 (2024) 1008-1021 ª 2024 The Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.