1. FERONIA Is a Key Modulator of Brassinosteroid and Ethylene Responsiveness in Arabidopsis Hypocotyls 
Deslauriers Stephen D. , Larsen Paul B.  
Molecular Plant 
Volume 3, Issue 3, Pages (May 2010)
DOI: /mp/ssq015
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Phenotypic Analysis of a Loss-of-Function feronia Mutant.
An ethylene dose–response analysis was performed in which seedlings of Col-0 wt and the mutant were grown for 4 d in the dark in the presence of either 5 μM AgNO3, which is an ethylene perception inhibitor, or 5 μM AVG, which effectively limits ethylene biosynthesis, with the latter supplemented with increasing concentrations of ethylene ranging from 0 to 100 μl L−1. The hypocotyl lengths of seedlings from each treatment were subsequently measured.
(A) Top panel represents actual hypocotyl length.
(B) Middle panel shows relative hypocotyl length (length/length at 5 μM AgNO3).
(C) Bottom panel shows the ratio of mutant hypocotyl length to Col-0 wt hypocotyl length for each ethylene concentration, with (—) denoting the predicted ratio if the mutant was not hyper-responsive to ethylene. Mean ± SE values were determined from 30 seedlings.
(D) Photographs of 4-day-old Col-0 wt and mutant dark-grown seedlings in the presence of 5 μM AgNO3, air, or saturating ethylene.
(E) Analysis of ethylene-responsive root growth. Dark-grown seedlings of Col-0 wt and the mutant treated with either AgNO3 or increasing concentrations of ethylene were analyzed for root growth, with the mutant significantly longer than wt at levels of ethylene that are highly inhibitory to root growth. Mean ± SE values were determined from 30 seedlings.
(F) This panel shows relative root length (length/length at 5 μM AgNO3) for both Col-0 wt and the mutant.
(G) The rate of ethylene production by 4-day-old dark-grown seedlings was measured for both Col-0 wt and the mutant. Ethylene was collected for 18 h and levels were determined by gas chromatography, with production rates calculated based on tissue fresh weight. Mean ±  SE values were determined from five samples of 100 seedlings each.
(H) Determination of adult rosette size of Col-0 wt and the mutant. Rosettes of 4-week-old plants of Col-0 wt and the mutant were collected and the total leaf area from each adult plant was measured. Mean ± SE values were determined from 10 plants.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Analysis of Ethylene-Dependent Gene Expression in fer-2.
(A) Expression of ethylene-responsive genes was determined for leaves of Col-0 wt and our mutant. For this analysis, 4-week-old adult plants of Col-0 wt and our mutant were exposed to air (A), 1 μl L−1 (LE), or 100 μl L−1 (HE) for 24 h, after which 10 μg of total RNA isolated from leaves was used for Northern analysis to test expression of the ethylene-responsive genes ERF1, ETR2, chiB, and PDF1.2. Tomato 18S rDNA was used to judge loading accuracy.
(B) Ethylene-responsive gene expression was determined in dark-grown seedlings of Col-0 wt and our mutant. For this analysis, Col-0 wt and mutant seedlings were grown in the presence or absence of 5 μM AgNO3 (Ag), air (A), 1 μl L−1 (LE), or 100 μl L−1 (HE) ethylene in the dark for 4 d, after which 10 μg of isolated total RNA was used for Northern analysis to test expression of genes known to be ethylene-responsive in seedlings including ACO2 and AtEBP. Tomato 18S rDNA was used to judge loading accuracy.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
A T-DNA Insertion in FERONIA Leads to Enhanced Ethylene Response in Arabidopsis.
(A) A mapping population was generated from a cross of our mutant (Col-0 background) to Ws-2 wt, following which F2 progeny that displayed the mutant phenotype in the presence of 10 μl L−1 ethylene were selected. Our mutation was localized to the bottom half of Arabidopsis chromosome 3 in a narrow genetic window between the polymorphic markers ALS and nga6. Thick bars represent the order of bacterial artificial chromosomes in the 18.8–19.2-Mb region of chromosome 3. Sequence analysis of candidate genes in this window resulted in the identification of a tandem insertion of the activation-tagging vector, pSKI15, in At3g51550, which encodes the receptor-like kinase, FERONIA. The genomic structure of FERONIA is depicted, including the position of our T-DNA insertion (fer-2) and a second T-DNA insertion (fer-3) identified using a reverse genetic approach.
(B) Functional complementation of the fer-2 mutation. A genomic construct of At3g51550 consisting of the promoter, 5′UTR, coding sequence, and 3′ UTR was introduced into the fer-2 mutant by Agrobacterium-mediated transformation. Subsequently, dark-grown Col-0 wt, fer-2, and T2 progeny from fer-2 transformed with the wt FERONIA genomic construct were tested for enhanced ethylene responsiveness after 4 d of growth with 1 μl L−1 ethylene.
(C) Analysis of the fer-3 allele, which represents a different T-DNA insertion in At3g51550, gives an identical phenotype compared to fer-2. Col-0 wt, fer-2, and fer-3 seedlings were grown in the dark in air for 4 d, after which hypocotyl length was assessed for each.
(D) Northern analysis of FERONIA expression in response to ethylene shows that it is ethylene-inducible. Total RNA was isolated from dark-grown Col-0 wt and fer-2 seedlings treated with 5 μM AgNO3 (Ag), air (A), 1 μl L−1 (LE), or 100 μl L−1 (HE) ethylene for 4 d, following which 10 μg of total RNA from each was used for Northern analysis with either FERONIA or tomato 18S rDNA, the latter of which was used to judge loading accuracy.
(E) FER expression is not brassinosteroid-responsive. Seedlings of Col-0 wt were grown in the light in either the absence or presence of 1 mM EBL, after which total RNA was isolated from each sample for Northern analysis with either FER or 18S rDNA, the latter of which was used to assess loading accuracy.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Genetic Analysis of the Relationship of fer-2 to Ethylene Signaling.
(A) A ctr1-3;fer-2 double mutant grown with an ethylene perception inhibitor has the extreme ethylene response normally seen for ethylene-treated fer-2, indicating that FERONIA functions at or downstream of CTR1. A ctr1-3;fer-2 double mutant was generated and compared to Col-0 wt, fer-2, and ctr1-3 with regard to its phenotype following growth for 4 d in the dark in the presence of 5 μM AgNO3.
(B) The etr1-1 mutation blocks the severe enhanced ethylene-response phenotype seen for fer-2. An etr1-1;fer-2 double mutant was generated and compared to Col-0 wt, etr1-1, and fer-2 with regard to its phenotype following growth for 4 d in the dark in the presence of 100 μl L−1 ethylene.
(C) The fer-2 mutation partially restores ethylene responsiveness to ein2-5, likely by amplifying the contribution of an EIN2 bypass to ethylene-dependent hypocotyl shortening. An ein2-5;fer-2 double mutant was generated and compared to seedlings of Col-0 wt, ein2-5, and fer-2 with regard to growth in the dark in the presence of 5 μM AgNO3, 10 μl L−1 ethylene, or 100 μl L−1 ethylene for 4 d. Hypocotyl lengths of each line were subsequently measured to quantify the level of ethylene response in ein2-5;fer-2. Mean ± SE values were determined from 30 seedlings. The photograph is of seedlings treated with 100 μl L−1 ethylene, showing the severe effects of ethylene on the overall growth of ein2-5;fer-2. t-tests were performed by comparing ethylene-treated samples to the Ag-treated sample of the same line. An asterisk indicates that a significant difference was found with P < 0.01.
(D) The fer-2 mutation restores ethylene response to hypocotyls of the ein3-1 mutant. An ein3-1;fer-2 double mutant was generated and analyzed for its ethylene responsiveness in comparison to Col-0 wt, fer-2, and ein3-1. For this analysis, seedling lines were grown for 4 d in the dark in the presence of 100 μl L−1 ethylene.
(E) An ein3-1;eil1-1;fer-2 is only slightly ethylene-responsive, likely due to the requirement of ethylene-dependent transcription for manifestation of a long-term response to ethylene. An ein3-1;eil1-1;fer-2 triple mutant was generated and analyzed in comparison to Col-0 wt, fer-2, and ein3-1;eil1-1 for its capability to respond to 100 μl L−1 ethylene following 4 d of growth in the dark.
(F) The reduced adult rosette size of fer-2 is not reversible by mutations that block ethylene signaling. Total leaf area was determined for untreated 4-week-old adult plants of Col-0 wt, fer-2, etr1-1, etrl-1;fer-2, ein2-5, ein2-5;fer-2, ein3-1;eil1-1, and ein3-1;eil1-1;fer-2. Mean ± SE values were determined from 10 plants.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Loss of feronia Results in Increased Responsiveness to Brassinosteroids in Light-Grown Seedlings.
(A) Hypocotyls of light-grown fer-2 seedlings are hyper-responsive to brassinosteroids. Seedlings of Col-0 wt and fer-2 were grown for 7 d in the light in the presence of 0, 0.1, or 1 μM 24-epibrassinolide (EBL), after which hypocotyl lengths were measured. Mean ± SE values were determined from 30 seedlings.
(B) Brassinosteroid-dependent hypocotyl elongation in light-grown seedlings is in part an ethylene-dependent phenomenon. Light-grown seedlings of Col-0 wt and fer-2 were treated with either 0 or 0.1 μM EBL in either the presence or absence of 5 μM AgNO3, after which hypocotyl lengths were determined. Mean ± SE values were determined from 30 seedlings.
(C) Mutations that confer ethylene insensitivity significantly block brassinosteroid-induced hypocotyl elongation of light-grown seedlings, including the brassinosteroid hyper-response seen for fer-2. Seedlings of Col-0 wt, fer-2, etr1-1, etr1-1;fer-2, ein2-5, and ein2-5;fer-2 were grown for 7 d in the light in either the absence or presence of 0.1 μM EBL, after which hypocotyls were measured. Mean ± SE values were determined from 30 seedlings.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Dark-Grown Hypocotyls of fer-2 Have Reduced Brassinosteroid Responsiveness.
(A) Hypocotyls of dark-grown fer-2 are hypersensitive to an inhibitor of brassinosteroid biosynthesis, BRZ. Col-0 wt and fer-2 were grown for 4 d in the dark in the presence of 5 μM AgNO3 and increasing concentrations of BRZ, after which hypocotyl length was determined. AgNO3 was included in this analysis in order to restore hypocotyl elongation of fer-2 to wt levels in the absence of BRZ. While Col-0 wt and fer-2 responded similarly to low concentrations of BRZ, treatment with higher concentrations resulted in a greater inhibitory effect on fer-2 hypocotyl growth compared to Col-0 wt.
(B) Hypocotyls of dark-grown fer-2 are partially brassinosteroid-insensitive. For this analysis, Col-0 wt and fer-2 were grown for 4 d in the dark in the presence of 5 μM AgNO3, 1 μM BRZ, and increasing concentrations of EBL, after which hypocotyl length was determined. While treatment with EBL largely restored hypocotyl elongation for Col-0 wt, fer-2 was found to be significantly less responsive to the complete range of EBL concentrations tested. Mean ± SE values were determined from 30 seedlings.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Brassinosteroid-Responsive Hypocotyl Shortening in the Dark Is Ethylene-Dependent.
Seedlings of Col-0 wt were grown in the presence of 5 μM AgNO3, 0.1 μM EBL, or 5 μM AgNO  μM EBL, while seedlings of ein2-5 were grown in either the absence or presence of 0.1 μM EBL for 4 d in the dark, after which hypocotyl lengths were measured. Mean ± SE values were determined from 30 seedlings.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Brassinosteroid-Responsive Shortening of Etiolated Hypocotyls Is Strictly Ethylene-Dependent.
(A, B) Dose–response analysis testing the effect of increasing concentrations of 24-epibrassinolide on hypocotyl elongation was performed. For this analysis, hypocotyl lengths of ein2-5 or Col-0 wt seedlings that were untreated or grown in the presence of either 2 μM AVG or 5 μM AgNO3 were determined following 4 d of growth in the dark. The top panel represents actual hypocotyl length, whereas the bottom panel represents relative hypocotyl length. Mean ± SE values were determined for 30 seedlings.
(C) For the EBL dose–response analysis, the number of seedlings at each EBL concentration that presented an apical hook was determined as a proportion of the total number of seedlings.
(D) In conjunction with the analysis of the effect of EBL on hypocotyl inhibition, the rate of ethylene production at various EBL concentrations in either the presence or absence of 2 μM AVG was determined. For this analysis, five samples comprising 100 seedlings each were measured for each treatment, with the presented data representing the mean ± SE.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
Model for the Role of Brassinosteroids and Ethylene in Control of Hypocotyl Elongation in Etiolated Seedlings.
(A) In wild-type etiolated seedlings, a balance exists between ethylene signaling, which promotes hypocotyl shortening, and FERONIA-mediated brassinosteroid signaling, which promotes hypocotyl elongation. In this model, an increase in the impact of one of the hormones, either through an increase in hormone concentration or a genetic perturbation affecting the opposing signaling pathway, will cause a change in this balance and lead to promotion of either hypocotyl shortening or elongation, depending on the hormone.
(B) A loss-of-function feronia mutant has exaggerated ethylene responsiveness due to loss of brassinosteroid responsiveness, which leads to a greater impact of ethylene on hypocotyl growth inhibition, since there is a loss of the contribution of brassinosteroids to this balance.
Molecular Plant 2010 3, DOI: ( /mp/ssq015)
Copyright © 2010 The Authors. All rights reserved. Terms and Conditions