SAUR63 stimulates cell growth at the plasma membrane

In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth

Primers used [Dataset]

1 table.S3 Table. Primers used.Peer reviewe

Localization of SAUR63:YFP:HA variants in Nicotiana benthamiana leaf cells

1 figure.A-L) Confocal images showing YFP fluorescence of indicated SAUR63:YFP:HA variants expressed in transiently transformed N. benthamiana leaves. Scale bar, 20 μm.Peer reviewe

Effect of mutating the SAUR63 clade

A) Genomic map showing positions of genes and locations of mutations from CRISPR/Cas9 mutagenesis in the 9x-saur mutant based on the TAIR10 Arabidopsis genome annotation. The first sgRNA directed cuts in both SAUR61 and SAUR64 (green arrows), creating a deletion between them (green bar) and leaving behind a hybrid gene with a frameshift at the junction site (symbolized by a green X). The second sgRNA directed cuts in the remaining genes (blue arrows, with lighter blue indicating slight mismatches between the sgRNA and the genome), leading to deletions (blue bars) and/or frameshift mutations (blue X’s). SAUR gene names are abbreviated as S61 etc. SAUR61-SAUR68 are on chromosome 1 and SAUR75 is on chromosome 5. B,C) 5-day-old seedlings grown on 1x MS/1% Suc medium in long days. Scale bar, 1 mm. D) Hypocotyl lengths of seedlings grown for 4d in short days on 0.5x MS medium. n, 27 (wild type), 22 (9x-saur). E) Cotyledon area of seedlings grown on vertically oriented plates for 6d on MS/1% Suc medium. n, 16 (wild type), 22 (9x-saur). Graphs show means ± s.d. No statistical differences were detected between wild-type and 9x-saur mutant measurements by t-test. F) Sequences of guide RNAs used for CRISPR/Cas9-mediated mutagenesis, wild-type genes, and mutant alleles present in the 9x-saur mutant. Underlines indicate PAM motif adjacent to guide RNA target site, and any mismatches to the guide RNA sequence. Uppercase bold letters indicate insertion mutations. All alleles create frameshift mutations except for saur75-1, which has an in-frame deletion of 13 amino acids in the SAUR domain.Peer reviewe

Seedling phenotypes of plants expressing SAUR63:YFP:HA variants

A) Cotyledon area of seedlings of indicated genotypes grown on vertically oriented plates for 7d in long days in the absence (open bars) or presence (filled bars) of 1% sucrose. B) Root length of seedlings of indicated genotypes grown on vertically oriented plates for 4d in long days in the absence (open bars) or presence (filled bars) of 1% sucrose. C,F) Hypocotyl lengths (E) and hypocotyl tortuosity index [F, 1 –(distance between ends)/(contour length)] of seedlings grown on vertically oriented plates for 3d in darkness without sucrose. D) Cotyledon areas of P35S:SAUR63 and P35S:CBL11-12:SAUR6326-142 seedlings grown for 6d on vertically oriented MS 1% Suc plates. E) Hypocotyl lengths of P35S:SAUR63 and P35S:CBL11-12:SAUR6326-142 seedlings grown for 4d on vertically oriented 0.5x MS plates. Panels D and E show data for the three homozygous single-locus P35S:SAUR63 lines that differed most from wild type among seven lines analyzed. Graphs show means ± s.d. Letters in graphs indicate statistical classes based on Tukey’s Honestly Significant Difference test. n, from left to right: panel A: 26, 20, 25, 15, 22, 23, 24, 20, 22, 23, 19, 19, 21, 19, 21, 20; panel B: 19, 16, 18, 14, 20, 20, 21, 16, 21, 17, 19, 16, 18, 15, 16, 17; panel D: 77, 18, 17, 17, 19, 20; panel E: 119, 25, 24, 27, 21, 24; panels C and F: 35, 36, 45, 43, 47, 38, 41, 47.Peer reviewe

NAAIRS mutants of SAUR63 and root tortuosity index

1 table.S2 Table. NAAIRS mutants of SAUR63 and root tortuosity index.Peer reviewe

Localization of SAUR63:YFP:HA in root meristem epidermal cells compared to control WAVE lines

A-D)P35S:SAUR63:YFP:HA. E-H)P35S:SAUR6326-142:YFP:HA. I-L)PUBQ10:WAVE 138Y expressing a PM-localized YFP fusion protein. M-P) PUBQ10:WAVE 1Y expressing a cytoplasmically-localized YFP fusion protein. Q-T) PUBQ10:WAVE 9Y expressing a YFP fusion protein localized to the tonoplast. Shown are fluorescence confocal microscopy images of YFP (green, A,E,I,M,Q), FM4-64 membrane staining (magenta, B,F,J,N,R), and both channels together (C,G,K,O,S) with vertical yellow lines indicating locations of quantitation of fluorescence intensity signals, scaled to the maximum signal along the line (D,H,L,P,T). Image color channel brightnesses were adjusted for visibility. Scale bar, 20 μm.Peer reviewe

Seedling phenotypes of plants expressing SAUR63:X fusion proteins

A,B) Hypocotyl length (A) and hypocotyl tortuosity index [B, 1 –(distance between ends)/(contour length)]) of seedlings of indicated genotypes grown for 3d in darkness on plates with 0.5x MS medium. C,D) Root length (C) and root tortuosity index (D) of seedlings of indicated genotypes grown for 4d in long days on plates with 1x MS 1% Sucrose medium. E,F) Root length (E) and root tortuosity index (F) of seedlings of indicated genotypes grown for 4d in short days on plates with 0.5x MS medium. Graphs show means ± s.d. Letters in graphs indicate statistical classes based on Tukey’s Honestly Significant Difference test. n from left to right: Panels A,B: 51, 24, 26, 28, 23, 25, 25; Panels C,D: 34, 17, 23, 14, 19, 15, 16; Panels E,F: 43, 21, 18, 17, 16, 15, 17. The same genotypes were measured in panels A-F, with genotype designations shown only in panels E and F. G,H,I) Hypocotyl epidermal cells of seedlings of indicated genotypes grown in short days for 2 days, visualized with the ML1:RFP shoot epidermis plasma membrane marker. Shown are z-stack confocal image projections of the near side of the hypocotyl. Scale bar, 0.1 mm. S2 Fig shows measurements of cell sizes in this experiment.Peer reviewe

SAUR63 fusion protein lipid binding

1 figure.A) Western blot showing presence of fusion proteins in extracts used in lipid blot experiments in Fig 5A. Arrows indicate locations of full-length SAUR63:YFP:HA and truncated SAUR6326-142:YFP:HA fusion proteins (upper arrow) and SAUR631-25:YFP:HA N-terminal domain fusion protein (lower arrow). B) Longer exposures of two lipid blots from Fig 5A. C) Mock experiment in which extracts were incubated for 70 minutes in protein extraction buffer at the indicated temperatures, and then run on a gel for western blots. For both SAUR63:YFP:HA and SAUR6326-142:YFP:HA, similar amounts of protein are present after incubation at -20 C or after incubation at 22 C, as during the lipid blot binding experiment.Peer reviewe

PEST:SAUR63NAAIRS:CerFP:HA lines

1 figure.A)PEST:SAUR63NAAIRS:CerFP:HA lines grown on plates with estradiol (all genotypes) or without estradiol (wild-type Columbia and PEST:SAUR63:CerFP:HA only). S2 Table indicates amino acids changed in each mutant and root tortuosity index measurement data from this and one replicate experiment. Scale bar, 1 cm. B) Western blots of total protein in estradiol-induced PEST:SAUR63NAAIRS:CerFP:HA lines using α-HA antibody.Peer reviewe