An increase of NPY1 expression leads to inhibitory phosphorylation of PIN-FORMED (PIN) proteins and suppression of pinoid (pid) null mutants
- Department of Cell and Developmental Biology, University of California San Diego, United States
Figures
Figure 1 with 1 supplement
A complete deletion of one copy of the PIN1 gene rescues pid null mutants.
(A) PIN1 gene is deleted using CRISPR/Cas9 gene editing technology. The two guide RNA target sites are shown with PAM underlined and in bold. The PIN1 start codon ATG is highlighted in yellow. The sequences flanking the deletions are shown. The two mutants have less than 18 bp of PIN1 coding sequence and are unlikely to produce any PIN1 proteins. (B) Heterozygous pin1 full #70, which had a deletion of 3078 bp of the PIN1 gene, restored the fertility of pid-TD1, a T-DNA insertion mutant that is completely sterile in wild-type (WT) PIN1 background. (C) pin1 full #32, when heterozygous, rescued pid-c1, a null pid allele.
Figure 1—figure supplement 1
Genetic interactions between pid and npy mutants.
(A) The phenotypes of pid-TD1 are not rescued by heterozygous npy1-2, which is a T-DNA insertion null allele. (B) The npy1-2 mutant is enhanced by heterozygous pid-TD1 mutant. Plants with npy1-2 pid-TD1+/- genotype often make pin-like inflorescences (arrow).
Figure 2 with 4 supplements
Overexpression of NPY1 rescues pid null mutants and triggers phosphorylation of PIN proteins.
(A) The NPY1 overexpression (OE) line #68 rescues the pid-c1, a null allele. Note that pid-c1 makes pin-like inflorescences and is completely sterile. pid-c1 no longer makes pin-like structures and is completely fertile when NPY1 protein levels are increased. A second NPY1 overexpression line (#83) also restored the fertility of pid-c1 (Figure 2—figure supplement 1). (B) NPY1 protein levels in the NPY1 OE lines in wild-type (WT) background and in pid mutants. In line #68, NPY1 protein level is 8.0-fold higher than WT, while line #83 has 9.7-fold higher NPY1 protein level than WT. In the absence of PID, overexpression of NPY1 leads to slightly lower NPY1 protein levels than the same transgenic event in WT background. (C) Overexpression of NPY1 leads to an increase in phospho-peptides. The volcano plot shows the fold changes (Log2 scale) of phospho-peptide levels in NPY1 OE lines over WT. Data above the horizontal dotted line are statistically significant. The vertical dotted line marks 2-fold change. (D) Overexpression of NPY1 leads to phosphorylation of PIN proteins. Among the phospho-peptides that displayed the most differences between NPY1 OE lines and WT are peptides from NPY1 and PIN proteins. In fact, in the upper-right quadrant in C, the only peptides were from NPY1 and PIN proteins.
Figure 2—figure supplement 1
Overexpression of NPY1 rescues pid mutants and causes obvious developmental phenotypes.
(A) Young adult NPY1 OE plants in wild-type (WT) and in pid-c1. The WT control was at the left. Note that the NPY1 OE plants had short petioles in both WT and in pid-c1 backgrounds. (B) NPY1 OE plants had more compact inflorescences and shorter stature compared to WT. (C) NPY1 OE completely eliminated pin structures in pid-c1 and developed normal looking inflorescences head. (D) Dissected flowers of WT and NPY1 OE plants. Note that flowers of NPY1 OE in pid-c1 occasionally have minor defects such as fewer stamens and fused organs (arrow).
Figure 2—figure supplement 2
Overexpression of NPY1 is sufficient to rescue pid null mutants.
(A) Overexpression of NPY1 rescues pid-TD1, which harbors a T-DNA insertion in the second exon and which is a strong allele. (B) Another T-DNA pid mutant, which also has the T-DNA insertion in the second exon, is rescued by overexpression of NPY1. (C) A close-up of the panel B. Some of the rescued pid-TD2 siliques only have one valve (arrows).
Figure 2—figure supplement 3
The identified phosphorylation sites in NPY1.
The NPY protein sequences along with NPH3 protein were aligned using ClustalW. Top panel shows the phosphorylation site S181 in NPY1. S181 is located in a highly conserved WSYT motif and is located in the region that separates the BTB domain and NPH3 domain. Middle panel shows a phosphorylation site inside the NPH3 domain. The bottom panel shows the predominant phospho-peptide ANHSPVASVAASSHSPVEK, which is located immediately after the NPH3 domain. Although the serine residues appear not to be conserved among NPY proteins, this region is rich in Ser/Thr residues in other NPY proteins and in NPH3. Previous studies have identified two phosphorylation sites in NPY1: S514 and S553, which are marked green. S553 was also identified as a phosphorylation site in this study (underlined green). S514 containing peptide was identified in this study, but it was S523 that was phosphorylated. The C-terminal ‘SIS’ motif in NPY1 also exists in NPH3. Previous studies have shown that the two serine residues in ‘SIS’ (in bold) in NPH3 are phosphorylated.
Figure 2—figure supplement 4
Overexpression of NPY1 leads to increases in phosphorylation of PIN proteins.
The hydrophilic loops of the ‘long PINs’ (PIN1,2,3,4,7) were aligned using ClustalW. The previously characterized phosphorylation sites are in bold black. The phosphorylated residues identified in NPY1 overexpression (OE) lines are highlighted in bold red. In the NPY1 overexpression lines, 10 phospho-peptides of PIN3 were identified, and 9 of them were significantly increased in the NPY1 OE lines compared to WT. Seven PIN7 phospho-peptides were identified, but only the peptide with S238 was significantly increased in NPY1 OE lines. More than a 4-fold increase in phosphorylation at S405 of PIN4 in NPY1 OE lines was observed. Phosphorylation of two highly conserved serine residues in PIN1 was increased in the NPY1 overexpression lines.
Figure 3 with 2 supplements
Overexpression of the truncated NPY1 lacking the C-terminal 30 amino acid residues (NPY1∆C) leads to smaller plants, agravitropic roots, and auxin resistance.
(A) Overexpression of NPY1∆C leads to small plant stature. The NPY1∆C plants (two independent lines, #35 and #4) have short petiole and epinastic leaves. (B) NPY1∆C plants have smaller flowers and are slower in developing siliques. (C) NPY1∆C #35 is much shorter than wild type (WT). NPY1∆C plants often have small pin-like structures (arrow). (D) A pin-like structure (arrow) of NPY1∆C #35 plants. (E) NPY1∆C plants have lost normal gravitropic responses. (F) NPY1∆C plants are auxin-resistant. Top panel: 5-day-old seedlings were transferred to MS media containing 100 nM 2,4-dichlorophenoxyacetic acid (2,4-D) and the root tips were marked. Bottom panel: The same plants from the top panel have grown for 3 more days. Note that roots of WT plants stopped growing, while roots of NPY1∆C plants continued to grow. 2,4-D did not rescue the defects of gravitropic responses. Plants from left to right: two NPY1∆C #4, two WT, two WT, two NPY1∆C #35.
Figure 3—figure supplement 1
Potential roles of the C-terminal domain of NPY1.
(A) The C-terminal domain (104 amino acid residues), which is located immediately after the conserved NPH3 domain, is rich in Ser/Thr (in bold). The C-terminal fragment (30 amino acid residues) contains 50% of Ser. The NPY1∆C construct deleted the C-terminal 30 amino acid residues (highlighted in red). (B) Potential PID-NPY1 interaction predicted from AlphaFold3. The PID (gold)-NPY1(blue)-ATP multimer predicted by the AlphaFold3 web server is shown. The disordered C-terminal tail of NPY1 was predicted to associate with the PID active site (red box). (C) Close-up of the predicted association between the NPY1 C-terminal tail and the PID kinase active site. Note the proximity of ATP, the catalytic aspartate D205 of PID, and S569, a possible phosphorylation target in NPY1 (see panel A). NPH3 contains a similar C-terminal motif to NPY1, and phosphorylation of the S569 counterpart in NPH3 (S744) by PHOT1 was shown to be involved in light signaling (https://doi.org/10.1038/s41467-021-26333-5).
Figure 3—figure supplement 2
Overexpression of NPY1∆C was not able to suppress pid-TD1.
Overexpression of NPY1∆C in pid-TD1 resulted in a smaller plant with pin-like inflorescences.
Figure 4 with 1 supplement
Overexpression of the truncated NPY1 lacking the C-terminal 30 amino acid residues (NPY1∆C) increases phosphorylation of PIN2 and other PINs.
(A) NPY1∆C levels are up several folds in two independent NPY1∆C overexpression lines (line #4 and line #35). (B) Overexpression of NPY1∆C led to increases in phosphorylation of many proteins. The phospho-peptides that were at least 2-fold enriched in NPY1∆C OE lines compared to wild type (WT) and that are statistically significant are shown in the upper-right quadrant. (C) Among the top 50 enriched phospho-peptides in NPY1 ∆C OE lines compared to WT, many are from NPY1 (blue dots) and PIN proteins. (D) PIN2 phosphorylation increased by at least 2-fold in both line #4 and line #35 for the three detected PIN2 peptides. Peptide I is SESGGGGsGGGVGVGGQNK, peptide II refers to KGsDVEDGGPGPR, and peptide III is HGYTNsYGGAGAGPGGDVYSLQSSK.
Figure 4—figure supplement 1
Overexpression of NPY1 lacking the C-terminal 30 amino acid residues (NPY1∆C) leads to increases in phosphorylation of PIN proteins.
The hydrophilic loops of the ‘long PINs’ (PIN1,2,3,4,7) are shown. The previously characterized phosphorylation sites are in bold and black. The phosphorylated residues identified in NPY1 OE lines, but not identified in the NPY1∆C OE lines, are highlighted in bold and yellow. The phosphorylated residues identified only in the NPY1∆C OE lines are highlighted in bold and green. Residues highlighted in purple and bold are identified in both NPY1 OE lines and the NPY1∆C lines.
Figure 5
The flower initiation pathway and phototropic pathway use analogous signaling mechanisms.
(A) Plasma-membrane-localized phototropins perceive blue light, causing changes in phosphorylation status of NPH3 and NPH3-PHOT association. In the nucleus, transcription factor ARF7/NPH4 also plays a role. (B) Pathway for auxin-mediated flower initiation. Three genes (PID, NPY, and MP) are homologous to their counterparts in phototropism (color-coded). The dotted arrows indicate that there are gaps in our understanding of the step. PHOT: PHOTOTROPIN; NPH3: NON-PHOTOTROPICAL HYPOCOTYL 3; ARF7: AUXIN RESPONSE FACTOR 7; MP: MONOPTEROS.
Tables
Table 1
Overexpression of NPY1 leads to increases in phosphorylation of PIN proteins.
NPY1 in wild-type (WT) 68 refers to NPY1 overexpression (OE) line #68 in WT background, while WT68 refers to plants without the NPY1 OE construct segregated from the NPY1 OE line #68. NPY1 in pid68 refers to the NPY1 OE line #68 in the pid-c1 homozygous background. The comparisons were made on the basis of the same transgenic event of integrating the NPY1 OE construct into the Arabidopsis genome. Similar nomenclature is used for line #83. Fold change over 1.5 is highlighted in orange, and p-value less than 0.05 is highlighted in green.
| Peptide | PINs | NPY1 in WT68/WT68 | NPY1 in WT83/WT83 | Average | NPY1 in WT/WT p-value | NPY1 in pid68/NPY1 in WT 68 | NPY1 in pid83/NPY1 in WT 83 | Average | NPY1 in pid/NPY1 in WT p-value |
|---|---|---|---|---|---|---|---|---|---|
| PSNLTGAEIYsLNTTPR | PIN7 | 0.9107672 | 0.8547737 | 0.88277044 | 0.012766228 | 1.112598883 | 1.158728114 | 1.1356635 | 0.033037985 |
| EAIEtGETVPVK | PIN7 | 0.927103551 | 0.8535734 | 0.89033847 | 0.070471392 | 1.055246666 | 1.046953254 | 1.05109996 | 0.080702772 |
| PSNLTGAEIYSLNTtPR | PIN7 | 0.951786878 | 0.8990926 | 0.92543972 | 0.406237963 | 1.12496421 | 1.003909907 | 1.06443706 | 0.294078921 |
| EAIEtGEtVPVK | PIN7 | 0.975410734 | 0.9260354 | 0.95072305 | 0.63905558 | 1.138387443 | 0.979226399 | 1.05880692 | 0.712820505 |
| AGLQVDNGANEQVGKsDQGGAK | PIN7 | 5.515410181 | 6.6516818 | 6.08354598 | 0.004312416 | 1.310119296 | 0.864392545 | 1.08725592 | 0.720644418 |
| ELHMFVWGsNGSPVSDR | PIN7 | 0.918218562 | 0.9385298 | 0.92837416 | 0.030446159 | 0.959554749 | 1.049222568 | 1.00438866 | 0.929513229 |
| DVNTNQQtTLPTGGK | PIN3 | 1.428745278 | 1.2713532 | 1.35004922 | 0.011613307 | 0.885671261 | 0.94839079 | 0.91703103 | 0.044539619 |
| MLVPDQsHNGETK | PIN3 | 1.758001858 | 1.8387322 | 1.79836702 | 0.001422119 | 0.87216277 | 0.927266836 | 0.8997148 | 0.067653426 |
| ELHMFVWsSNGSPVSDR | PIN3 | 0.956359302 | 1.0992576 | 1.02780847 | 0.626786946 | 1.075031196 | 1.060510928 | 1.06777106 | 0.077051066 |
| AVAHPASGDFGGEQQFsFAGK | PIN3 | 1.664595268 | 1.5428397 | 1.6037175 | 0.003053126 | 0.916818466 | 0.931886419 | 0.92435244 | 0.079198469 |
| DVNTNQQTTLPTGGKsNSHDAK | PIN3 | 1.206078414 | 1.2145015 | 1.21028997 | 0.005949912 | 0.829404603 | 1.001475074 | 0.91543984 | 0.237603568 |
| LAPNSTAALQsK | PIN3 | 1.615052147 | 1.2956924 | 1.4553723 | 0.014269456 | 0.888615931 | 1.022036706 | 0.95532632 | 0.340591893 |
| AVAHPAsGDFGGEQQFsFAGK | PIN3 | 2.028214914 | 1.8376356 | 1.93292524 | 0.002815952 | 1.003513383 | 0.866937161 | 0.93522527 | 0.4099271 |
| AVAHPAsGDFGGEQQFSFAGK | PIN3 | 3.252097884 | 4.7317962 | 3.99194703 | 0.000937954 | 1.092715151 | 0.864714055 | 0.9787146 | 0.776844974 |
| AGLNVFGGAPDNDQGGRsDQGAK | PIN3 | 5.672290509 | 6.3210041 | 5.99664729 | 0.004496914 | 1.217637228 | 0.777071055 | 0.99735414 | 0.92826952 |
| DVNTNQQTTLPTGGKSNsHDAK | PIN3 | 1.305382555 | 1.1061201 | 1.20575132 | 0.171206898 | 0.867146124 | 1.185891699 | 1.02651891 | 0.965006443 |
| KsGGDDIGGLDSGEGER | PIN4 | 1.042670534 | 1.4020681 | 1.2223693 | 0.12829056 | 1.042840596 | 0.974466076 | 1.00865334 | 0.853312205 |
| GGGDDIGGLDsGEGER | PIN4 | 3.710114314 | 5.0168001 | 4.36345721 | 0.002196075 | 1.073696602 | 0.907341208 | 0.9905189 | 0.886638269 |
| NSNFGPGEAVFGsK | PIN1 | 1.520973667 | 1.2937862 | 1.40737993 | 0.00749093 | 0.964169889 | 0.963473082 | 0.96382149 | 0.041626442 |
| NsNFGPGEAVFGSK | PIN1 | 1.154637794 | 1.2017641 | 1.17820094 | 0.005035937 | 0.959448057 | 0.890138014 | 0.92479304 | 0.079103255 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis) | NPY1 | NCBI | Gene ID: 829311 | Also called ENP |
| Gene (Arabidopsis) | PID | NCBI | Gene ID: 818030 | |
| Strain, strain background (Arabidopsis) | NPY1 overexpression line 68 in wild-type Arabidopsis | This paper | NPY1 OE in WT 68 | Available from this group upon request |
| Strain, strain background (Arabidopsis) | Wild-type Arabidopsis line segregated out from NPY1 overexpression line 68 | This paper | WT 68 | Available from this group upon request |
| Strain, strain background (Arabidopsis) | NPY1 overexpression line 68 in pid Arabidopsis | This paper | NPY1 OE in pid 68 | Available from this group upon request |
| Strain, strain background (Arabidopsis) | NPY1 overexpression line 83 in wild-type Arabidopsis | This paper | NPY1 OE in WT 83 | Available from this group upon request |
| Strain, strain background (Arabidopsis) | Wild-type Arabidopsis line segregated out from NPY1 overexpression line 83 | This paper | WT 83 | Available from this group upon request |
| Strain, strain background (Arabidopsis) | NPY1 overexpression line 83 in pid Arabidopsis | This paper | NPY1 OE in pid 83 | Available from this group upon request |
| Genetic reagent (Arabidopsis) | pid knockout generated by CRISPR | PMCID: PMC10691239 | pid-c1 | Available from this group upon request |
| Genetic reagent (Arabidopsis) | pid-TD1 mutant (Arabidopsis) | PMCID: PMC10691239 | pid-TD1 | Available from this group upon request |
| Genetic reagent (Arabidopsis) | pid-TD2 mutant (Arabidopsis) | PMCID: PMC10691239 | pid-TD2 | Available from this group upon request |
| Transfected construct (Arabidopsis) | plasmid | This paper | UBQ-NPY1 | Available from this group upon request |
Additional files
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Supplementary file 1
Phospho-peptides from NPY1 protein in both wild-type (WT) and pid-c1 backgrounds.
NPY1 overexpression T1 plants with pid-c1+/- genotype was self-pollinated. At the T2 stage, three genotypes were selected: WT without transgenes and without pid-c1 mutation (this is called WT-xx, xx refers to the line number); NPY1 OE without pid-c1 mutation (called NPY1 in WT xx); NPY1 OE in pid c1 (called NPY1 in pid xx). Inflorescence heads with flower buds of the lines were used for proteomic analysis. All of the detected NPY1 peptides were more abundant in the overexpression lines than in WT. The two most abundant peptides in NPY1 OE lines were highlighted in yellow. PID did not affect NPY1 phosphorylation, as all of the NPY1 phospho-peptides were detected in pid-c1 background.
- https://cdn.elifesciences.org/articles/108207/elife-108207-supp1-v1.docx
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Supplementary file 2
Phosphorylation of NPY1 and PIN proteins caused by NPY1∆C overexpression.
L4 and L35 refer to NPY1∆C OE lines #4 and #35, respectively. WT: wild type. Lower case s or t in the peptides refers to the phosphorylated residues. The PIN2 phospho-peptides are highlighted in yellow, which were enriched in the NPY1∆C OE lines.
- https://cdn.elifesciences.org/articles/108207/elife-108207-supp2-v1.docx
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Supplementary file 3
Primers used in this study.
- https://cdn.elifesciences.org/articles/108207/elife-108207-supp3-v1.docx
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Supplementary file 4
TMT labeling schemes.
- https://cdn.elifesciences.org/articles/108207/elife-108207-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108207/elife-108207-mdarchecklist1-v1.docx
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An increase of NPY1 expression leads to inhibitory phosphorylation of PIN-FORMED (PIN) proteins and suppression of pinoid (pid) null mutants
eLife 14:RP108207.
https://doi.org/10.7554/eLife.108207.2
