Abstract
The developmental plasticity of the root system plays an essential role in the adaptation of plants to the environment. Among many other signals, auxin and its directional, intercellular transport are critical in regulating root growth and development. In particular, the PIN-FORMED2 (PIN2) auxin exporter acts as a key regulator of root gravitropic growth. Multiple regulators have been reported to be involved in PIN2-mediated root growth; however, our information remains incomplete. Here, we identified ROWY Bro1-domain proteins as important regulators of PIN2 sorting control. Genetic analysis revealed that Arabidopsis rowy1 single mutants and higher-order rowy1 rowy2 rowy3 triple mutants presented a wavy root growth phenotype. Cell biological experiments revealed that ROWY1 and PIN2 colocalized to the apical side of the plasma membrane in the root epidermis and that ROWYs are required for correct PM targeting of PIN2. In addition, ROWYs also affected PIN3 protein abundance in the stele, suggesting the potential involvement of additional PIN transporters as well as other proteins. A global transcriptome analysis revealed that ROWY genes are involved in the Fe2+ availability perception pathway. This work establishes ROWYs as important novel regulators of root gravitropic growth by connecting micronutrient availability to the proper subcellular targeting of PIN auxin transporters.
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Introduction
Land plants utilize the root system to support themselves and to absorb water and nutrients from the soil. Root growth and development require the coordination of internal signals and environmental cues and exhibit highly adaptive plasticity under different environmental conditions. The phytohormone auxin (with the major natural form being indole-3-acetic acid (IAA)) and the related molecular framework play fundamental roles in almost every aspect of plant life, including root growth and developmental regulation1. Genetic studies have established the molecular network for auxin biosynthesis2,3,4, transport5,6, and signaling7,8, which cooperate in a multitude of auxin functions.
For IAA biosynthesis, the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED PROTEIN (TAR) and YUCCA (YUC) enzymes catalyze the conversion of tryptophan to indole-3-pyruvic acid (IPyA) and subsequently IAA, which is a major pathway in plants2,9,10,11. In addition, multiple modifications of IAA, such as oxidation or conjugation to amino acids, control IAA homeostasis in plants2. The nuclear TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALLING F-BOX (AFB)-AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) pathway mediates auxin function in transcriptional regulation7,12 and in nontranscriptional rapid responses related to the regulation of root growth13,14,15. Recently, studies have revealed that cell-surface AUXIN BINDING PROTEIN1 (ABP1)-TRANSMEMBRANE KINASE (TMK)-based auxin perception results in rapid cellular auxin responses through global modulation of the phosphoproteome16.
Directional intercellular auxin transport, namely, polar auxin transport (PAT), is a key feature of auxin action. The plasma membrane (PM)-localized PIN-FORMED (PIN) family of auxin transporters pump auxin out of the cell, playing essential roles in PAT17,18,19,20,21. Intriguingly, PIN transporters reside asymmetrically at the PM, and this polarity of PIN proteins determines the directionality of intercellular auxin flow, which is crucial in many patterning processes as well as in asymmetric growth during tropic responses5,6,22. ABC transporters have also been reported to be involved in auxin export23,24, but recent work has suggested that ABCB19 is a major brassinosteroid transporter25. Moreover, AUXIN1 (AUX1)/LIKE AUXINs (LAXs) are auxin importers26. In addition, intracellular auxin transporters, such as PIN-LIKES (PILS), tonoplast-associated WALLS ARE THIN1 (WAT1), and TOB1 (TRANSPORTER OF IBA1), potentially control free auxin homeostasis via subcellular compartmentalization inside the cell5,6.
There are eight members of the PIN family of transporters: PIN1 ~ PIN4 and PIN7 localize to the PM, and PIN5, PIN6, and PIN8 reside in the ER (with PIN6 also at the PM), with the latter participating in intracellular auxin transport5,27. Among those PIN transporters, PIN2 (also known as AGRAVITROPIC1/AGR1, ETHYLENE INSENSITIVE ROOT1/EIR1 and WAV6) controls root growth and development28, and it is expressed mainly in the epidermis, cortex, and lateral root cap in the root meristem. Loss-of-function pin2 mutants exhibit agravitropic roots, and a critical function for correct plasma membrane targeting and the intracellular distribution of PIN2 for directional root growth is underlined by characterization of trans-acting regulators. AGC [named cAMP-dependent (PKA) and cGMP-dependent protein kinase (PKG) and protein kinase C (PKC)] kinases, such as PINOID (PID) and WAVY ROOT GROWTHs (WAGs), regulate the polarity and activity of the PIN2 protein by phosphorylating its cytosolic loop29,30,31. The BTB family E3 ligases MACCHI-BOU4 (MAB4)/ENHANCER OF PID (ENP)/NAKED PINS IN YUCCA-LIKE1 (NPY1) protein and homologous MELs (MAB4/ENP/NPY1-LIKEs) are essential for the establishment and maintenance of apical PIN2 polarity in the root epidermis31,32. Similarly, recent studies imply that WAVY GROWTH3 (WAV3) and WAV3-LIKE E3 ligases are critical regulators of PIN2 apical polarity and thus root gravitropism22,33.
Multiple hormonal and nutrient signaling pathways converge on PIN2 to regulate growth6,34, including the cytokinin35, ethylene22,28, brassinosteroid36,37, salicylic acid38,39,40,41,42, abscisic acid43,44, jasmonic acid45, strigolactone46, and mTOR and nitrogen signaling pathways47. In addition, auxin itself also regulates PIN PM localization, forming a feedback loop controlling PIN abundance at the PM16,48,49,50.
In this study, we revealed that Arabidopsis Bro1 domain proteins, ROOT WAVY GROWTHs (ROWYs), modulate root growth and development by positively regulating PIN2 stability and subcellular dynamics. Specifically, our work provides novel insights into PIN-mediated polar auxin transport and related root patterning processes and links PIN2 function to variations in micronutrient availability.
Results
Coexpression analysis identified ROWY1 as a putative regulatory gene that acts in conjunction with PIN2 in Arabidopsis roots
PIN2 is a key regulator of root growth and development22,28,31. To identify putative regulatory genes involved in PIN2-mediated root growth and development, we performed a coexpression analysis in the ATTED-II (http://atted.jp) database (Fig. 1a). Multiple coexpressed genes, such as CNGC14 and BEARSKIN1/2 (Fig. 1a), were previously reported to be involved in root development or gravitropism51. AT5G14020 (hereafter named ROOT WAVY GROWTH1, ROWY1, previously also named BRo1-domain protein As FREE1 suppressor, BRAF) encodes a Bro1-domain protein, which was previously identified as a regulator of the ESCRT-I-mediated sorting of plasma membrane cargo via competitively binding to the VPS23 subunit52. However, the biological functions of ROWY1 have not been reported previously.
In the Arabidopsis genome, there are three genes encoding ROWY1 and its closest homologs: AT5G14020 (ROWY1), AT1G17940 (ROWY2), and AT1G73390 (ROWY3, previously also named AtBro1)53 (Supplementary Figs. 1 and 2). Phylogenetic analysis revealed that ROWYs are homologs of ALIX (Supplementary Figs. 1 and 3), which is a critical regulator of the ESCRT pathway in eukaryotes. The available single-cell RNA-seq data indicated that ROWY1, ROWY2, and ROWY3 were expressed in root cells (Supplementary Fig. 4). To further study the expression patterns of the ROWY genes, we constructed promoter-driven GUS reporter lines. GUS staining of the resulting lines revealed that ROWY1 ~ ROWY3 were expressed in multiple tissues (Supplementary Fig. 5), and specifically, they were detected in root meristems (Fig. 1b, c). Notably, although all three ROWY genes are expressed in the root, GUS staining revealed some differences in the expressed cell layers compared with the single-cell RNA-seq data, which also reflects the requirement for cell biological verification for high-throughput data. In contrast to the constitutive pattern observed from the single-cell RNA-seq data, ROWY3 was predominantly expressed in root columella cells and lateral root cap cells, whereas ROWY2 was expressed in the division and elongation zones. Together with the results from the transcriptomics analysis, these findings indicate that ROWY functions in root meristems.
The rowy1 rowy2 rowy3 triple mutant results in a wavy growth root phenotype in Arabidopsis
To study the biological functions of the ROWY genes, T-DNA insertional mutants, rowy1-1, rowy1-2, rowy2-1, rowy2-2, rowy3-1, and rowy3-2, were obtained and verified via PCR analysis (Supplementary Fig. 6a, b). Further RT‒qPCR and RT‒PCR analyses revealed that rowy1-1, rowy1-2, rowy2-1, and rowy2-2 were knockout mutants, whereas rowy3-1 and rowy3-2 appeared to represent knockdown alleles (Supplementary Fig. 7a, b).
Phenotypic analysis revealed that rowy1-1 and rowy1-2 single mutants presented increased root waviness when grown on vertically oriented nutrient plates, whereas rowy2-1, rowy2-2, rowy3-1, and rowy3-2 did not (Supplementary Fig. 8a–c). Double and triple mutants were generated by crossing, and a more pronounced root wavy growth phenotype was observed for the rowy1-1 rowy2-2 rowy3-2 (hereafter referred to as rowy123, used for further analyses) and rowy1-2 rowy2-1 rowy3-1 triple mutants, suggesting functional redundancy of the ROWY genes (Fig. 2a–f, Supplementary Fig. 8d–f, and Supplementary Fig. 9). Specifically, the primary root of rowy123 was longer than that of wild-type Col-0 (Fig. 2c), and the root tip angles of rowy123 were more variable than those of wild-type Col-0 (Fig. 2e, f). Consistent with the wavy root phenotype, rowy123 mutants presented a smaller root gravitropic index value (distance/primary root length) than did Col-0 (Fig. 2c, d, and Supplementary Fig. 9). To study the role of ROWY genes in the root gravitropic response in more detail, vertically grown seedlings were turned 90°, followed by determination of the kinetics of root tip reorientation over time. This analysis revealed that the roots of rowy123 bent more rapidly than those of Col-0 did (Supplementary Fig. 10). Notably, the wavy root growth phenotype of rowy123 plants was also observed in the dark (Supplementary Fig. 11). Therefore, we speculated that the wavy root growth phenotype of rowy123 might be due to both defects in gravitropic growth and its faster growth rate. Notably, the gravitropic phenotype of rowy123 mutants is different from that of pin2 mutants, which usually bend randomly under gravistimulation conditions. We speculate that the wavy root phenotype of rowy123 might be due to a defect in bending termination during gravitropism.
To further analyze the expression and function of the ROWY genes in wavy root growth, the transgenic lines rowy123 pROWY1::ROWY1-GFP and rowy123 pROWY2::ROWY2-GFP were generated. Analysis of the root phenotype at the seedling stage revealed that the pROWY1::ROWY1-GFP transgene rescued the root phenotype of rowy123 in terms of the wavy phenotype (Supplementary Fig. 12a–d), whereas pROWY2::ROWY2-GFP did not (Supplementary Fig. 12e–h). This finding is consistent with the fact that the rowy1 mutants presented a root wavy phenotype, whereas the rowy2 and rowy2 rowy3 mutants did not, suggesting a predominant role of ROWY1 in controlling root growth.
The ROWY1 protein is localized to the apical plasma membrane domain in root epidermal cells in Arabidopsis
To study the subcellular localization of ROWY1, a complemented rowy123 pROWY1::ROWY1-YFP transgenic line was used for analysis. Imaging via confocal laser scanning microscopy (CLSM) revealed that ROWY1-YFP is expressed mainly in the epidermis, lateral root cap, and columella cells in the root meristem (Fig. 3a). Notably, both ROWY1-YFP and ROWY1-GFP exhibited polar subcellular localization enriched at the apical side of the root meristem epidermis cells (Fig. 3a, b). A comparison of the signals from the pROWY1::ROWY1-GFP and pROWY2::ROWY2-GFP lines with those from the pPIN2::PIN2-GFP (eir1-4) line in root meristem epidermis cells as a control revealed striking overlap, with predominant enrichment of reporter signals at the apical domain of the plasma membrane (PM) (Fig. 3b, c and Supplementary Fig. 13).
It was reported previously52 that ROWY1/BRAF showed dual localization at both the PM and MVB/LE/PVC, and the PM targeting of ROWY1 might be due to S-acylation of cysteine 3 (C3). Consistently, treatment with the S-acylation inhibitor 2-BP (2-bromopalmitate) abolished the PM localization of ROWY1-GFP, suggesting that S-acylation might indeed be essential for ROWY1 PM targeting (Supplementary Fig. 14a). Further analysis of the pROWY1::rowy1C3A-GFP allele, with the presumptive C3 S-acylation site substituted by alanine (A), demonstrated that mutant rowy1C3A-GFP failed to localize to the PM, confirming the importance of the C3 residue for ROWY1 subcellular localization (Supplementary Fig. 14a). Remarkably, the transgene harboring rowy1C3A-GFP rescued the wavy root phenotype of rowy123 but failed to rescue rowy123 root elongation defects, suggesting that discernible subcellular ROWY1 localization impacts distinct aspects of rowy123 root growth defects (Supplementary Fig. 14b–d). Given that the wild-type version of the pROWY1::ROWY1-GFP transgene partially rescued the root elongation phenotype of rowy123 (Supplementary Fig. 12a–d), we speculate that PM targeting is required for its full function. In line with observations suggesting that ROWY1 functions as a negative regulator of the ESCRT complex at MVB/LE/PVC52, we speculate that both the PM and MVB/LE/PVC subcellular localizations of ROWY1 contribute to its role in controlling root growth.
Involvement of ROWY genes in the regulation of auxin-mediated root growth in Arabidopsis
To examine the functional relationship between ROWY1 and PIN2, a pROWY1::ROWY1-YFP pPIN2::PIN2-mCherry (eir1-4) line was generated. Imaging via CLSM revealed that the ROWY1-YFP and PIN2-mCherry signals strongly overlapped at the apical plasma membrane domain of the root epidermis cells, which is consistent with regulatory crosstalk between these proteins (Fig. 4a, b). Notably, ROWY1-YFP was also expressed in and localized to the apical side of the cortex layer in the elongation zone but not in the division zone, where PIN2, however, localized to the basal side. These findings suggest that ROWY1 might have additional functions in root development. To explore the genetic interaction between ROWYs and PIN2, a cross was made for rowy123 and eir1-4 (also named pin2-T). The root phenotype of rowy123 eir1-4 revealed epistasis of eir1-4 to rowy123, suggesting that the ROWYs function upstream of PIN2 (Fig. 4c–e). Consistently, pharmacological treatments with salicylic acid (SA), ibuprofen (IBU), TIBA or NPA, compounds that interfere with polar auxin transport, enhanced the agravitropic root phenotype of rowy123, substantiating a function for ROWYs upstream of elements of the auxin transport machinery (Supplementary Fig. 15).
We next introduced the pPIN2::PIN2-Venus (eir1-4) reporter into rowy123 to test the consequences of a loss of ROWY genes on PIN2 reporter fate. Notably, the signal intensity of pPIN2::PIN2-Venus (eir1-4) was markedly lower in rowy123 than in the wild-type controls (Fig. 4f–i). The assessment of PIN2-Venus signal distribution in the root meristem epidermis cells revealed more intracellular signals in rowy123 than in the wild type (Fig. 4h–j), which is indicative of deficiencies in PIN2 intracellular trafficking. Brefeldin A (BFA) treatment predominantly affects the plasma membrane targeting of PINs by interfering with steps in the secretory pathway and with protein recycling between the plasma membrane and the endomembrane system16,48,49,50. As a result, BFA causes the accumulation of cargo proteins in intracellular so-called BFA bodies. While there was less PIN2-GFP in rowy123 at the PM, the BFA body in rowy123 was smaller than that in the WT (Supplementary Fig. 16a, b), suggesting a defect in the intracellular trafficking of the PIN2 cargo protein. Further treatment with the PI3K inhibitor wortmannin (Wm) also revealed a similar intracellular distribution of the PIN2 cargo protein (Supplementary Fig. 16c, d). In addition, the deficiency of ROWYs led to a decrease in the abundance of PIN2 at the PM in both epidermal cells and cortical cells in the elongation zone, and a similar decrease was also observed in lateral root cap (LRC) cells (Supplementary Fig. 17a–f).
Further analysis of pPIN3::PIN3-GFP revealed that there was a decrease in the PIN3-GFP signal in the stele in rowy1-1 rowy3-2, whereas the PIN3-GFP fluorescence in the columella cells of the root cap in rowy1-1 rowy3-2 was similar to that of the WT (Supplementary Fig. 18a, b). In addition, there was no obvious change in pPIN7::PIN7-GFP in rowy123 (Supplementary Fig. 18c, d). Therefore, we speculate that ROWY genes might be involved in the endomembrane trafficking of a subset of PIN cargo proteins, among which PIN2 shows major changes. The decrease in PIN3 protein and possibly additional PIN members in the stele might contribute to rootward auxin transport and thus auxin accumulation in the root tip. Moreover, whether columellar cell-expressed PIN members might also play roles in the auxin stream from the root cap to the elongation zone requires further investigation.
Given the essential roles of PIN2 in mediating both auxin flux from the root tip to the root elongation zone and auxin reflux to the root tip, we asked whether exogenously applied auxin could rescue root growth deficiencies that arise as a consequence of impaired PIN2 distribution in rowy123. Indeed, auxin treatments rescued the wavy growth phenotype of rowy123 (Fig. 5a–c), suggesting that altered auxin homeostasis resulting from loss of the ROWY genes could contribute to rowy123 phenotypes. To further test this hypothesis, we introduced a DR5rev::GFP auxin-responsive reporter into rowy123 by crossing. Quantitative CLSM analysis revealed a decrease in DR5 signal intensity in rowy123, which was partially rescued by external auxin application (Fig. 5d, e). These results suggest that the wavy root growth phenotype of rowy123 might be due to lower auxin levels or a lower capability to maintain maximal auxin, potentially arising as a consequence of alterations in PIN2- and additional PINs-mediated directional auxin transport. Further analysis of the DR5rev::GFP reporter in the root gravitropic response by 90° reorientation revealed a decrease in DR5 relocation at 4 h, suggesting a defect in shootward auxin transport (Supplementary Fig. 19). This is in line with the decreased PIN2 levels in the PM in the lateral root cap cells.
The Fe2+ deficiency-responsive pathway regulates root wavy growth in Arabidopsis
Our results suggest that ROWY genes play an essential role in regulating root growth and development in Arabidopsis. However, the upstream signaling of ROWY genes is still unknown. To test this hypothesis, an RNA-seq experiment was performed with Col-0 and rowy123 seedlings. Approximately 107 differentially expressed genes (DEGs) were identified in rowy123 compared with those in Col-0, of which 32 genes were upregulated and 75 genes were downregulated (Fig. 6a, b, and Supplementary Data 1). GO analysis suggested that multiple Fe deficiency-related genes were enriched in these DEGs. For example, VACUOLAR IRON TRANSPORTER-LIKE1 (AtVTL1), AtVTL2, and AtVTL554, which encode for proteins that catalyze Fe transport into vacuoles into vacuoles into vacuoles, were upregulated in rowy123 mutants. To further test whether Fe deficiency is involved in the regulation of root growth and development, Arabidopsis Col-0 and rowy123 seedlings were grown on +Fe2+ (normal MS media) and -Fe2+ media, respectively. The results revealed that Col-0 seedlings presented a wavy root phenotype on -Fe2+ MS media, similar to that of rowy123 plants on normal MS media (Fig. 6c–e). A growth assay of the gravitropic response revealed that Fe2+ deficiency led to faster gravitropic growth, which is similar to the phenotype of rowy123 (Supplementary Fig. 20a, b). Further analysis of PIN2::PIN2-Venus and the DR5rev::GFP auxin-responsive reporter revealed that Fe2+ deficiency led to a decrease in the PIN2 signal and an increase in the DR5 signal in the root tip (Fig. 6f, g, and Supplementary Fig. 20c, d). Therefore, we hypothesized that ROWY genes might be involved in iron-modulated root growth and development.
Discussion
Auxin plays essential roles in regulating plant growth and development. The root system architecture is largely shaped by various environmental cues and intrinsic signals, and auxin is a central signal. The root-waving phenotype is reportedly regulated by exogenous mechanical cues as well as endogenous hormones and proteins, such as the microtubule cytoskeleton55. Here, we revealed that the ROWY1 gene is a key regulator of root waving. Our data suggest that ROWY1, possibly together with other ROWY members, is involved in PIN2-mediated root gravitropic growth. In line with the enhanced wavy growth phenotype in the rowy123 triple mutant, there was a greater intracellular distribution of PIN2. Thus, we hypothesize that ROWYs are positive regulators of PIN2 polar PM targeting. However, the roots of rowy123 bent faster than did those of the WT, suggesting a potential regulatory mechanism for PIN2 protein homeostasis at the PM to avoid overbending. Notably, there was a decrease in DR5 redistribution in rowy123 during the root gravitropic response, suggesting the presence of additional regulatory layers for the maintenance of maximal auxin in the root tips. In addition, PIN3 expression decreased in the root stele, and the decreased DR5 signal could be a combination of both basipetal shootward auxin transport and rootward auxin transport in the root tips, suggesting the potential involvement of additional PIN auxin transporters.
ROWY1 is polarly distributed at the apical side of the PM in the root epidermis, where PIN2 also resides. The PM targeting of ROWY1 relies on its S-acylation at the C3 residue. However, disruption of S-acylation by amino acid substitution or chemical inhibition does not seem to be required for the biological function of ROWY1 in root growth. Together with the previous finding that ROWY1 might function as a negative regulator of the ESCRT complex at MVB/LE/PVC52, we speculate that PM recycling from MVB/LE/PVC might function as a negative regulatory mechanism for ROWY1. Whether this type of posttranslational modification is responsive to external or internal cues might be an interesting open question awaiting further investigation.
Iron is an important nutrient for plant growth and development56. Here, we revealed that Fe2+ availability is a key regulator of wavy root growth. Notably, multiple related genes were upregulated in the rowy123 triple mutant, and Fe2+ availability strikingly shaped the root system architecture, resembling rowy123. Notably, Fe2+ availability also modulates PIN2 subcellular distribution and related auxin distribution. Therefore, we propose that ROWY proteins are regulators of the Fe2+ homeostasis pathway. These data identify ROWYs as molecular hubs linking Fe nutrients to root growth, but the detailed molecular mechanisms involved require further research. A recent study revealed that ROWY3 (AtBro1) might be involved in ABA signaling and the abiotic stress response53. Interestingly, all three ROWY genes are predicted to have multiple splice variants, and whether alternative splicing plays a role in regulating ROWY3 function awaits further investigation.
Methods
Plant materials and growth conditions
Chlorine-sterilized Arabidopsis seeds were subsequently grown on ½ MS media supplemented with 1% (w/v) sucrose and 0.8% plant agar (pH 5.8). After 2 days of cold treatment (4 °C), the seeds were moved to a growth chamber at 22 °C under a 16-h light/8-h dark photoperiod. After 7 days of growth on MS media, the seedlings were transferred to soil and allowed to grow under the same conditions for 2–3 months until the plants matured.
Single mutants rowy1-1 (WiscDsLox412H04), rowy1-2 (GABI_041F11), rowy2-1 (SALK_035256), rowy2-2 (SALK_038492), rowy3-1 (SALK_204462C), and rowy3-2 (SALK_063092) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). The transfer DNA (T-DNA) insertion sites were verified via PCR. The fluorescent reporter lines pPIN1::PIN1-GFP 57, pPIN2::PIN2-GFP 58, pPIN2::PIN2-Venus37, pPIN2::PIN2-mCherry59, pBRAF1::BRAF1-YFP 52, pPIN3::PIN3-GFP 60, pPIN7::PIN7-GFP 60, and DR5rev::GFP 61 were reported previously. These reporters were introduced into the rowy123 triple mutant background by crossing. The floral dip method was used for Arabidopsis transformation with Agrobacterium tumefaciens strain GV3101. The fluorescent reporter lines and Promoter::GUS transgenic lines for ROWYs were generated via transformation. The mutants and transgenic lines used in this study are listed in Supplementary Table 1. All compounds are listed in Supplementary Table 2. The primers used for identifying T-DNA insertion sites and genotyping cross lines are listed in Supplementary Table 3.
Molecular cloning
Restriction enzyme digestion or Gateway cloning was used to make constructs for transgenic plant generation. For restriction enzyme digestion, the restriction enzyme sites were added to DNA fragments via PCR and then cloned and inserted into the corresponding plasmids. For Gateway cloning, DNA fragments were first cloned and inserted into entry vectors via the BP reaction and then cloned and inserted into destination vectors via the LR reaction. Point mutagenesis was performed with a QuikChange II Site-Directed Mutagenesis Kit (200524, Agilent Technologies). All reagents, including restriction enzymes and Gateway clonases, are listed in Supplementary Table 2. The primers used for molecular cloning are listed in Supplementary Table 3. The resulting plasmids from this study are listed in Supplementary Table 4.
Quantitative RT‒PCR (RT‒qPCR) analysis
RT‒qPCR was used to examine the transcripts of ROWY1 ~ ROWY3 in rowy mutants, with ACTIN7 (AT5G09810) as an internal reference. In detail, total RNA was extracted from the indicated tissues via TRIzol (Invitrogen), and DNase was added to digest the genomic DNA. For RT‒PCR, first-strand cDNA was generated via a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). PCR was performed to amplify the full-length CDSs of ROWY1, ROWY2 and ROWY3. For RT‒qPCR, 1 µg of each RNA sample was reverse transcribed (Takara). The resulting cDNA of the corresponding genes and ACTIN7 was analyzed via SYBR Premix Ex Taq (Takara) with a Bio-Rad CFX Connect Real-Time System. The relative transcript levels of the examined genes were normalized to those of ACTIN7, calculated by setting the wild-type or a certain tissue as “1”, and finally presented as the average ± standard deviation (s.d.) from three biological replicates. The primers used are listed in Supplementary Table 3.
Promoter::β-glucuronidase (GUS) histochemical staining
For the promoter‒GUS fusion studies, 1.5-kilobase (kb) genomic DNA fragments containing the promoter regions of the ROWY1, ROWY2, and ROWY3 genes were amplified via PCR and subcloned and inserted into a modified pCambia1300 binary vector62 (the primers used for molecular cloning are listed in Supplementary Table 3). Positive transgenic lines were used for staining.
The plant materials (seedlings, rosette leaves, inflorescences, fruits, etc.) were incubated in freshly prepared staining solution (100 mM Na-phosphate buffer, 0.1% (v/v) Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, pH 7.0) supplemented with 0.5 mg/ml X-Gluc at 37 °C for 2‒3 h. Then, the stained materials were decolorized with 90% and 70% ethanol at room temperature until the chlorophyll was completely removed. Specifically, for the imaging of roots, the decolorized materials were mounted with a transparent liquid (chloral hydrate:H2O:glycerol = 8:3:1).
Immunostaining
Four-day-old roots were immunostained as previously described48. Seedlings were fixed with PBS supplemented with 4% paraformaldehyde (PFA) for 1 h under vacuum. The seedlings were washed three times with PBS containing 0.1% Triton for 10 minutes, followed by washing three times with water containing 0.1% Triton for 10 min. After incubation with 2% driselase in PBS at 37 °C for 1 h, the seedlings were washed three times with PBS containing 0.1% Triton and then incubated with PBS supplemented with 10% DMSO and 3% NP40 at room temperature for 1 h. Seedlings were washed three times with PBS supplemented with 0.1% Triton for 10 min, followed by blocking with 2% bovine serum albumin (BSA) in PBS at room temperature for 1 h. Subsequently, the seedlings were incubated with a-GFP antibody (Sigma, 1:1000) in blocking solution at 37 °C for 4 h, followed by washing five times with PBS supplemented with 0.1% Triton for 10 min and incubation with Cy5-a-mouse antibody (1:600) in blocking solution at 37 °C for 4 h. The seedlings were washed three times with PBS containing 0.1% Triton for 10 min, followed by washing three times with water containing 0.1% Triton for 10 minutes. The samples were mounted in nonhardening mounting medium and imaged with an LSM 800 inverted confocal laser scanning microscope (Zeiss).
Microscopy and image acquisition
For confocal laser-scanning microscopy (CLSM), a Zeiss LSM980 instrument equipped with a GaAsP detector (Zeiss) was used. Various confocal settings were set to record the emission of different fluorophores: EGFP (excitation, 488 nm; emission, 507 nm), YFP (excitation, 488 nm; emission, 527 nm), Venus (excitation, 488 nm; emission, 529 nm), mCherry (excitation, 543 nm; emission, 610 nm), mRFP (excitation, 543 nm; emission, 607 nm), and PI (excitation, 495 nm; emission, 635 nm). All of the images were obtained at an 8-bit depth with 2× line averaging.
The GUS-stained samples were imaged via a DIC microscope (Olympus BX53).
Phenotypic analysis
Col-0, rowy1-1, rowy1-2, rowy2-1, rowy2-2 single mutants, rowy1 rowy2, rowy2 rowy3, rowy1 rowy3 double mutants and rowy1 rowy2 rowy3 triple mutants were uniformly seeded on 1/2 MS media. After 2 days of cold treatment (4 °C), the seeds were moved to the growth chamber, and the other conditions were completely consistent. After a certain number (as indicated in the figure legends) of days, MS plates were imaged with a Sony A6000 camera with a macro lens, and then the primary root length, growth distance, or root tip angles were analyzed with ImageJ software.
Pharmacological treatments and experimental conditions
For exogenous auxin treatments, Arabidopsis seeds were sown on MS plates supplemented with the indicated exogenous auxin, including IAA (Sigma), NPA (Sigma), and 2,4-D (Sigma). After stratification for 2 d at 4 °C, the plates were transferred to the growth chamber under the conditions described in the “Plant materials and growth conditions” section for 6 d according to different assays. The phenotype was then analyzed via ImageJ.
For short-term treatment to study the subcellular localization of PIN2::PIN2-Venus and PIN2::PIN2-Venus in rowy123, 4-day-old seedlings grown on normal MS plates were transferred to 100 μM BFA or 33 μM Wm liquid MS for 1 h. Afterwards, the samples were imaged via CLSM.
Accession numbers
The sequence data from this study can be found in the Arabidopsis Genome Initiative or enBank/EMBL databases. The accession numbers are as follows: ROWY1 (AT5G14020), ROWY2 (AT1G17940), ROWY3 (AT1G73390), PIN1 (AT1G73590), PIN2 (AT5G57090), PIN3 (AT1G70940), and PIN7 (AT1G23080).
RNA-seq analysis
For RNA-seq analysis, the roots were cut from 7-day-old Col-0 and rowy123 seedlings. Four independent RNA samples (100 mg) were used for the following analyses. RNA extraction and RNA-seq were performed by BGI via the BGISEQ platform. The reads of each sample were aligned to the publicly available reference genome of Arabidopsis (TAIR10, https://support.illumina.com/sequencing/sequencing_software/igenome.html) via HISAT2 version 2.0.4 with default parameters. Data analyses were performed with the Dr. Tom platform of BGI. The differentially expressed genes of Col-0 and rowy123 with log2 > 0 are listed in Supplementary Data 1. GO enrichment analysis was based on the classification of the GO_P term description as log2 > 0. P values were calculated via the phyper function, and Q values were obtained via FDR correction of P values. Functions with a Q value < = 0.05 are considered significantly enriched. Heatmap analysis was performed via OECloud tools (https://cloud.oebiotech.com) as log2 > = 0.58.
Statistics and reproducibility
Most experiments were repeated at least three times independently, with similar results obtained. The results are reported as the means ± SD. To measure primary root length, growth distance, and root-tip angles, photographs were analyzed via ImageJ (https://imagej.nih.gov/ij/download.html). The fluorescence intensity of the CLSM images was quantified via Fiji (https://fiji.sc/). Data visualization and statistical analysis were mostly performed with GraphPad Prism 8. For the bending curvatures of the roots, polar bar graphs were generated via Origin 2021. The n value represents the number of seedlings or independent experiments. P values were calculated with a two-tailed t test for quantitative variables between two independent groups or one-way ANOVA for quantitative variables among three or more independent groups. The results were considered statistically significant when the p value was less than 0.05.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
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Acknowledgements
We thank Drs. Erika Isono (University of Constance), Grégory Vert (University of Toulouse), and Liwen Jiang (The Chinese University of Hong Kong) for kindly sharing published Arabidopsis lines; Dr. Yuzhou Zhang (ISTA) for help with molecular cloning, and Drs. Melinda Abas (BOKU), Eugenia Russinova (Ghent University), and Zhaojun Ding (Shandong University) for valuable discussions. This work was supported by grants to S.T. from the National Natural Science Foundation of China (32321001), the USTC Research Funds of the Double First-Class Initiative (YD9100002016), the Research Funds from the Center for Advanced Interdisciplinary Science and Biomedicine of IHM, the Division of Life Sciences and Medicine, the University of Science and Technology of China (QYPY20220012), the Fundamental Research Funds for the Central Universities (WK9100000021), and start-up funding from the University of Science and Technology of China and the Chinese Academy of Sciences (GG9100007007, KY9100000026, KY9100000051, and KJ2070000079). J.S. was supported by the National Natural Science Foundation of China (31970181 and 32170342). J.F. was supported by Austrian Science Fund (FWF; projects I6123 and P37051-B).
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S.T. conceived the project and designed the experiments. The project was initiated by S.T. in the J.F. lab. Y.P., K.J., Y.M., and Y.W. performed the physiological and cellular experiments under supervision of S.T., J.F., and E.B. All the authors contributed to the data analysis. B.K., C.L., and J.S. contributed reporter lines and provided critical suggestions. Y.P., J.F., and S.T. wrote the manuscript with input from other coauthors, and all authors revised and approved the submitted version.
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Peng, Y., Ji, K., Mao, Y. et al. Polarly localized Bro1 domain proteins regulate PIN-FORMED abundance and root gravitropic growth in Arabidopsis. Commun Biol 7, 1085 (2024). https://doi.org/10.1038/s42003-024-06747-9
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DOI: https://doi.org/10.1038/s42003-024-06747-9
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