1. Volume 7, Issue 8, Pages 1316-1328 (August 2014)
Arabidopsis RAN1 Mediates Seed Development through Its Parental Ratio by Affecting the Onset of Endosperm Cellularization 
Peiwei Liu, Ming Qi, Yuqian Wang, Mingqin Chang, Chang Liu, Mengxiang Sun, Weicai Yang, Haiyun Ren 
Molecular Plant 
Volume 7, Issue 8, Pages (August 2014)
DOI: /mp/ssu041
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
RAN1 Expression Levels in RAN1 T-DNA Mutants, Overexpression Lines, and Seed Phenotypes Caused by RAN1 Misexpression.
(A) Diagram of the RAN1 gene and insertion sites of T-DNA mutants r1-1 and r1-2. Gray boxes, exons; gray lines connecting gray boxes, introns; white box, untranslated region (UTR); black line, promoter; triangle, T-DNA insertion site.
(B) Expression levels of the RAN family in r1-2 and in wild-type, as determined by qRT–PCR analysis.
(C) Expression levels of RAN1, RAN2, and RAN3 in r1-2 and in wild-type, as determined by qRT–PCR analysis.
(D) Expression levels of RAN1 in T-DNA lines r1-1 and r1-2, complemented line pRAN1::R1COM, and the overexpression lines pRAN1::R1OX1, pRAN1::R1OX2, pRAN1::R1OX3, as determined by qRT–PCR analysis. qRT–PCR results were collected from 7-day-old plants. The means were calculated from three biological samples. Each biological sample was examined in quadruplicate. The calculated SE includes technical error and biological error.
(E) (a–f) Mature seeds from RAN1 T-DNA mutants and overexpression lines. (a) Wild-type. (b) r1-1. (c) r1-2. (d) pRAN1::R1OX1. (e) pRAN1::R1OX2. (f) pRAN1::R1OX3. (g)pRAN1::R1GFPCOM. (h)pRAN1::R1COM. Bar = 2mm. The number in the black box shows the average mass of 100 seeds.
The seed data are the means from 28 wild-type, 28 mutants, and 28 trans-genetic lines. The experiment was independently repeated three times. ** indicates statistically significant differences between values (Student's t-test, P < 0.01).
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Tissue Expression Pattern of RAN1 in Arabidopsis.
(A) GUS staining of pRAN1::GUS in Arabidopsis.(a) Seven-day seedlings. Bar = 1mm. (b) Flowers. Bar = 1mm. (c) Wild-type flowers (negative control). Bar = 1mm. (d)pRAN1::GUS in stamen. Bar = 0.5mm. Inset, pollen tube. Bar = 30 μm. (e) Pistil. Bar = 1mm. (f) Ovule. (g–k)pRAN1::GUS expressed in a developing seed. (g) Globular embryo stage. (h) Early heart embryo stage. (i) Late heart embryo stage. (j) Mature seed. (k) Mature embryo. Bars in (f–k) are 100 μm.
(B)RAN1 expression level in Arabidopsis. L, leaf; S, stem; F, flower bud; Si, silique.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Seed Development in Reciprocal Crosses between Wild-Type and RAN1 T-DNA Mutants.
(A) Mature F1 seeds from reciprocal crosses between wild-type and RAN1 T-DNA mutants. Bar = 2mm. (a)r1-1 crossed with wild-type. (b)r1-2 crossed with wild-type. (c)pRAN1::R1COM crossed with wild-type. (a') Wild-type crossed with r1-1. (b') Wild-type crossed with r1-2. (c') Wild-type crossed with pRAN1::R1COM. The number in the black box shows the average mass of 100 seeds.
(B) Developmental stages of F1 seeds from the reciprocal cross between wild-type and r1-2. Seeds were cleared and imaged with differential contrast optics at 3, 5, 6, 9, and 15 DAP. Bars = 50 μm. (a1–a5) Wild-type × wild-type. (b1–b5)r1-2 × wild-type. (c1–c5) Wild-type × r1-2. (d1–d5)r1-2 × r1-2.
The seed data are the means from 28 wild-type and 28 mutant lines. The experiment was independently repeated three times.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Subcellular Localization and Parent-Specific Expression Pattern of RAN1.
(A)RAN1–GFP subcellular localization. (a) Hypocotyl cells, (b) root cells, (c) pericycle cells, (d) whole ovule, (e) endosperm. (a'–e') Merged images. Bars = 20 μm.
(B) Expression level of RAN1 in wild-type and in pRAN1::R1GFPCOM, as determined by qRT–PCR analysis.
(C) RT–PCR and restriction analysis of cDNA from seeds dissected into endosperm + seed coat fractions and embryo fractions at an early developmental stage (3–5 DAP) and at a later developmental stage (8 DAP).
(D) Allele-specific sequencing of RAN1 cDNA from the endosperm + seed coat fraction at 4 and 8 DAP; embryos at 4 DAP. The biallelic-expressed maternal and paternal alleles show a double peak at the polymorphic nucleotide. EN+S, endosperm + seed coat; E, embryo.
(E) (a–e) Wild-type ovules pollinated with pRAN1::GUS pollen. (a) Twelve hours after hand pollination. Inset shows ovules. Bar = 50 μm. Black arrowhead shows a pollen tube of pRAN1::GUS (as paternal contributor) growing into wild-type micropyle. Bar = 100 μm. (b) 2 d, (c) 3 d, (d) 4 d, (e) 12 d seed after pollination. (a'–e')pRAN1::GUS ovules pollinated with wild-type pollen. (a') 12h, (b') 2 d, (c') 3 d, (d') 4 d, (e') 12 d seed after pollination. Bar = 100 μm. All pRAN1::GUS plants were homozygotes. At least 20 independently propagated wild-type or independent transgenic lines were analyzed. The experiment was repeated three times.
(F) (a) RAN1–GFP expression in the endosperm of F1 seeds from the wild-type crossed with pRAN1::R1GFPCOM on 3 DAP. (a') RAN1–GFP expression in the endosperm of F1 seeds from pRAN1::R1GFPCOM crossed with WT on 3 DAP. Bar = 20 μm.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Expression of RAN1 in the Endosperm of F1 Seeds from the Reciprocal Cross between Wild-Type and r1-2.
(A) Expression of RAN1 in the endosperm of F1 seeds from a wild-type self-cross.
(B)r1-2 self-cross.
(C) Wild-type × r1-2.
(D)r1-2 × wild-type. qRT–PCR results were collected in the endosperm + seed coat fractions during 2–7 DAP.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 Cellularization during Seed Development.
(A) (a–d) Wild-type self-cross. (a'–d') Bright fields.
(B) (a–d)r1-2 self-cross. (a'–d') Bright fields.
(C) (a–d) Wild-type × r1-2.(a'–d') Bright fields.
(D) (a–d)r1-2 × wild-type. (a'–d') Bright fields. The green fluorescence is generated by auto-inflorescence after treatment with glutaraldehyde (see the ‘Methods’ section). Red arrowheads show new cell walls in the endosperm. Light-red shapes show the shapes of embryos. Bar = 50 μm.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7 Genetic Relation of RAN1 and MINI3 in the Reciprocal Cross.
(A) (a) Mature seeds of a wild-type self-cross. (b) Homozygote r1-2 crossed with wild-type. (c) Homozygote r1-2 crossed with homozygote mini3. Bar = 5mm.
(B) Diagram of MINI3 gene and insertion sites in T-DNA mutants mini3. Gray boxes, exons; gray lines connecting gray boxes, introns; triangle, T-DNA insertion site. Primers for MINI3 expression identification are shown as arrows.
(C) qRT–PCR analysis of RAN1 relative expression levels in wild-type and mini3 seeds.
(D)MINI3 relative expression levels of F1 seeds from a reciprocal cross between wild-type and r1-2 as well as of F1 seeds from r1-2 crossed with mini3 at 5 DAP.
(E) qRT–PCR analysis of the relative expression levels of MINI3 in wild-type pollen and in mini3 pollen.
(F) Average mass of 100 mature F1 seeds from a wild-type self-cross, r1-2 crossed with wild-type, and from r1-2 crossed with mini3.
The seed data are the means ± SE from 28 wild-type and 28 mutant lines. The experiment was independently repeated three times. ** indicates P < 0.01.
Molecular Plant 2014 7, DOI: ( /mp/ssu041)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions