Research ArticlePLANT SCIENCES

Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants

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Science Advances  11 Dec 2020:
Vol. 6, no. 50, eabc8895
DOI: 10.1126/sciadv.abc8895
  • Fig. 1 Intraspecies genetic complementation analysis of Arabidopsis PIN function in its inflorescence/floral organ patterning.

    (A) Phylogenetic relationship of the homologous PIN members in the flowering plant A. thaliana. The canonical PINs are divided into three clades: PIN1, PIN2, and PIN3/4/7. (B) Phenotypical analysis of the Arabidopsis pin mutants with disruption of one or two clades of the canonical PINs (PIN1 and PIN3/4/7). Phenotypical analysis of the shoots with 7-week-old plants and the roots with 1-week-old seedlings. (C) The Arabidopsis null mutant pin1 showed severe defects in inflorescence/floral organ formation. Genetic complementation experiments with Arabidopsis paralogous PINs showing that of the A. thaliana canonical PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and noncanonical PINs (PIN5 and PIN6); all canonical PINs, but not noncanonical PINs, were able to complement the naked inflorescence of pin1 mutant. Only PIN1 was able to fully rescue the defective floral organ with no stamens and structurally aberrant petals. Phenotypic analysis of the shoot system with 7-week-old plants. (D) Anatomical structure of flower from the PIN transformants in (C). n = 100 flowers from each transgenic line. (E) Silique length and seed number per silique of PIN transformants in (C). n = 13 to 19 siliques from each transgenic line. Photo credit: Yuzhou Zhang, Institute of Science and Technology (IST) Austria.

  • Fig. 2 Intraspecies genetic complementation analysis of Arabidopsis homologous PIN function in its shoot/root patterning.

    (A) The Arabidopsis loss-of-function pin1/3/4/7 quadruple mutant showed severe defects with the arrested shoot and root development, and genetic complementation experiments with Arabidopsis canonical PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and noncanonical PINs (PIN5 and PIN6) showed that only the canonical PINs were able to rescue the severe shoot/root defects of Arabidopsis pin1/3/4/7 quadruple mutant. Phenotypic analysis of the shoots with 7-week-old plants and the roots with 1-week-old seedlings. (B to J) Auxin maximum in root tips of the PIN transformants in (A), indicated by the synthetic auxin-responsive reporter DR5rev::GFP in 7-day-old Arabidopsis seedlings. The level of DR5rev::GFP expression is reflected by signal intensity. The GFP channel images are shown in pseudocolor, and the intensity scale is shown at the left (H, high; L, low). The white arrowhead indicates the quiescent center. Scale bar, 20 μm. (K) Root lengths of wild type, pin1/3/4/7 mutant, and PIN transformants after growing for of 3, 5, and 7 days, respectively. Data represent means ± SD (n = 11 roots from each line). (L) Quantification of the DR5rev::GFP signal intensity in wild type, pin1/3/4/7 mutant, and PIN transgenic lines shown in (D) to (J). Data represent means ± SD (n = 6 roots from each line). Photo credit: Yuzhou Zhang, Institute of Science and Technology (IST) Austria.

  • Fig. 3 The divergence of both coding and cis-regulatory domains of Arabidopsis PIN members.

    (A) Schematic diagram showing three functions of Arabidopsis canonical PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and noncanonical PINs (PIN5 and PIN6) in plant patterning according to the evolutionarily functional analysis. (B) Schematic diagram of the generation of an auxin maximum in the root by the Arabidopsis PIN auxin transporters. (C and D) Immunolocalization showing that PIN1 is specifically expressed in the stele of Arabidopsis root with rootward (basal) subcellular localization (C), while PIN2 is exclusively expressed in the lateral sides of Arabidopsis root with shootward (apical) subcellular localization in epidermal cells (D). (E) In pin1;pPIN1::PIN2 transgenic lines, immunolocalization showing that the ectopic expression of AtPIN2 in the stele (middle of the root) under the control of PIN1 promoter results in its basal subcellular localization similar to that of AtPIN1 (C). The yellow arrowheads indicate the PIN polarity in root cells. Scale bar, 10 μm. (F) Function of chimeric PIN proteins (V1 and V2) with domains swapped between PIN1 and PIN2 in floral organ development. (G) Statistical analysis of the flower patterns of the transgenic lines carrying the chimeric PIN proteins V1 and V2 in (F). The transgenic lines with PIN1 or PIN2 expressed in Arabidopsis pin1 mutant background under the control of PIN1 promoter were used as the control. n = 100 flowers from each transgenic line. Photo credit: Yuzhou Zhang, Institute of Science and Technology (IST) Austria.

  • Fig. 4 PIN function in Arabidopsis shoot/root development originated at land plants.

    (A to I) The interspecies complementation experiments with homologous PIN genes from a green alga (KfPIN) (A), a marchantiophyte (MpPINZ) (B), a moss (PpPINA) (C), a lycophyte (SmPINR) (D), a gymnosperm (PtPINH and PtPINE) (E and F), and three flowering plants Amborella trichopoda (AmtPIN1a) (G), Arabidopsis (AtPIN1) (H), and C. rubella (CarPIN1) (I). Except for the green alga gene encoding KfPIN (A), all of the land plant canonical PIN genes were able to rescue the severe defects of pin1/3/4/7 mutant in shoot/root development (B to I). Phenotypic analysis of the shoots with 7-week-old plants and the roots with 1-week-old seedlings. (J to S) Auxin maximum in root tips of the 7-day-old Arabidopsis PIN transformants in (A) to (I), indicated by synthetic auxin-responsive reporter DR5rev::GFP. The level of DR5rev::GFP expression is reflected by signal intensity. The GFP channel images are shown in pseudocolor, and the intensity scale is shown at the left (H, high; L, low). The white arrowhead indicates the quiescent center. Scale bar, 20 μm. (T) Quantification of root lengths of wild type, pin1/3/4/7 mutant, and PIN transformants in (A) to (I) after growing for 3, 5, and 7 days, respectively. Data represent means ± SD (n = 11 roots from each transgenic line). (U) Quantification of the DR5rev::GFP signal intensity in wild type, pin1/3/4/7 mutant, and transgenic lines in (J) to (S). Data represent means ± SD (n = 6 roots from each transgenic line).

  • Fig. 5 PIN function in Arabidopsis floral organ development emerged at flowering plants.

    (A to I) The interspecies complementation experiments with homologous PIN genes from a green alga (KfPIN) (A), a marchantiophyte (MpPINZ) (B), a moss (PpPINA) (C), a lycophyte (SmPINR) (D), a gymnosperm (PtPINH and PtPINE) (E and F), and three flowering plants A. trichopoda (AmtPIN1) (G), Arabidopsis (AtPIN1) (H), and C. rubella (CarPIN1) (I). Only the flowering plant genes encoding AmtPIN1a, AtPIN1, and CarPIN1 from the PIN1 clade were able to rescue the Arabidopsis pin1 defects in floral organ formation and thus led to seed formation in the siliques (G to I). The canonical PINs from vascular plants (lycophyte SmPINR, gymnosperm PtPINE, and PtPINH) and flowering plants (AmtPIN1, AtPIN1, and CarPIN1) have the capacity to rescue the defective phenotype of Arabidopsis pin1 with naked inflorescence (D to I). Phenotypic analysis of the shoot system with 7-week-old plants. (J) Anatomical structure of the floral organ from PIN transformants in (A) to (I). n = 100 flowers from each transgenic line. (K) Statistical analysis of the flower pattern of PIN transformants in (A) to (I). (L) Silique length and seed number per silique concerning these PIN transformants in (A) to (I). n = 13 to 19 siliques from each transgenic line. (M) Phenotype of the inflorescence in weak allele pin1-5 mutant, null allele pin1 mutant, wild type, and pin1-5;pPIN1::AtPIN1 transgenic line. (N) Anatomical structure of the floral organ from wild type, pin1-5, and pin1-5;pPIN1::AtPIN1 transgenic line. (O) Statistical analysis of the flower pattern of the PIN transformants in pin1-5 mutant background. n = 100 flowers from each transgenic line. (P) Silique length and seed number per silique concerning these PIN transformants in the pin1-5 mutant background. n = 13 to 19 siliques from each transgenic line. Photo credit: Yuzhou Zhang, Institute of Science and Technology (IST) Austria.

  • Fig. 6 The polarity of heterologous PIN in Arabidopsis root stele cells and the contribution of stepwise functional innovations of the PIN protein to the origin of flowering plants.

    (A to G) Cellular polarity analysis of KfPIN (A), MpPINZ (B), PpPINA (C), SmPINR (D), PtPINE (E), PtPINH (F), and AtPIN1 (G) in Arabidopsis root stele cells under the control of Arabidopsis PIN1 promoter by fusion with GFP protein. The blue, yellow, and white arrowheads indicate the apical, basal, and lateral localization of PIN proteins in root stele cells, respectively. Scale bars, 10 μm. (H) Ratio of PIN-GFP intensity between the basal side and the designated lateral side toward the outside of the root. The blue arrowheads indicate the two cellular sides for analysis. Data represent means ± SD (n = 10 from each transgenic line). (I) Schematic showing the three-step functional innovation that occurred in the PIN protein during plant evolution. The three disparate functions of PIN (i.e., shoot/root development, inflorescence development, and floral organ formation), which evolved in three distinct plant evolution milestones (the origin of land plants, vascular plants, and flowering plants), are associated with the patterning and growth of flowering plants as exemplified by Arabidopsis, implying the indispensable contribution of PIN evolution to the origin of flowering plants.

Supplementary Materials

  • Supplementary Materials

    Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants

    Yuzhou Zhang, Lesia Rodriguez, Lanxin Li, Xixi Zhang, Jiří Friml

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