1. Volume 5, Issue 1, Pages 205-217 (January 2012)
A Novel Protein RLS1 with NB–ARM Domains Is Involved in Chloroplast Degradation during Leaf Senescence in Rice 
Jiao Bin-Bin , Wang Jian-Jun , Zhu Xu-Dong , Zeng Long-Jun , Li Qun , He Zu-Hua  
Molecular Plant 
Volume 5, Issue 1, Pages (January 2012)
DOI: /mp/ssr081
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Phenotypes of the Wild-Type and rls1 Mutant.
(A) Leaves phenotypes of 30-, 45-, and 70-day-old wild-type and rls1 mutant plants. Cell death lesions began to appear on 45-day-old rls1 leaves.
(B) TEM analysis of cells of the wild-type leaf and rls1 spotted leaf, indicating the destroyed chloroplasts in the rls1 cell in the lesion spot. c, chloroplast; v, vacuole.
(C) Naturally senescent leaves of the wild-type and rls1 at 25 d after flowering. F, the flag leaf; 2, the second leaf; 3, the third leaf.
(D) Matured wild-type and rls1 plants 45 d after flowering. Note that most parts of the rls1 plant were dead. Bar = 15 cm.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Physiological Changes in Wild-Type and rls1 Leaves during Dark-Induced Senescence.
(A) Wild-type and rls1 plants were grown for 3 weeks under 13/11 h light (30°C)/dark (28°C), and then transferred to the dark for 5 d to induce leaf senescence.
(B) Content of chlorophyll during dark incubation. Dotted lines, wild-type; solid lines, rls1. Error bars indicate standard deviations (SD), n = 6.
(C) Fv/Fm values. Dotted lines, wild-type; solid lines, rls1. Error bars indicate SD, n = 6.
(D) Membrane ion leakage. Dotted lines, wild-type; solid lines, rls1. Error bars indicate SD. n = 6.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Ultrastructures of Plastids in Wild-Type and rls1 Cells.
(A–C) The ultrastructures of the wild-type cells; (A) the cell of wild-type; (B) the chloroplast in the wild-type cell; (C) the mitochondrion in the wild-type cell.
(D–I) The ultrastructures of rls1 cells; (D) the cell of rls1; (E) the chloroplast in the cell of rls1; (F) the mitochondrion in the cell of rls1; (G) the spherical body in the chloroplast; (H) fusion of the spherical body with the vacuole; (I) degradation of the spherical body in the vacuole. c, chloroplast; g, grana stack; m, mitochondrion; p, plastoglobuli; s, starch granule; sp, spherical bodies; v, vacuole.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 Map-Based Cloning of RLS1.
(A) The RLS1 locus was mapped to a region between markers M5 and M2 on the short arm of rice chromosome 2.
(B) Fine-mapping of RLS1 to a 60-kb genomic DNA region between two makers M17 and M24. Two BAC contigs (AP and AP005008) cover the RLS1 locus.
(C) Sequence comparison revealed a substitution of C to T in the second exon of the gene LOC_Os02g10900 in the rls1 mutant.
(D–F) Genetic complementation of the rls1 mutant. (D) The leaves of the wild-type, the rls1 mutant, and one representative complemented line, photographs were taken 25 d after flowering. F, the flag leaf; 2, the second leaf; 3, the third leaf. (E) Plants of the wild-type, rls1, and the complemented line, photographs were taken 45 d after flowering. (F) Seed set of the wild-type, rls1, and the complemented line.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 Alignment of the NB Domains of RLS1 and Other Proteins.
(A) Alignment of the NB domain of RLS1 with NB domains of other proteins. The conserved motifs are indicated.
(B) The phylogenetic tree constructed based on alignment of the NB domain sequences of RLS1 with NB domains of other proteins. XP_ from sorghum, CBI3628 from grape, NC_ from black cottonwood, I2, R3a, Rx from potato, RRS1-R, CHS3 from Arabidopsis, N from tobacco, APAF1 from human, CED4 from C. elegans.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
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7. Figure 6 Expression Analysis of RLS1.
(A) Expression patterns of RLS1 in the leaf of seedling (LS), root of seedling (RS), flag leaf during heading stage (LH), leaf sheath (LS), young panicle (PH) and spikelet (SH) of the wild-type. Values are means ± SD; n = 3.
(B) RLS1 expression in leaves of 3-week-old wild-type plants after transferred to darkness. RNA was isolated at 0 (control), 4, 8, 12, 24, 48, and 72 h after being transferred to darkness. Values are means ± SD; n = 3.
(C) RLS1 expression was suppressed by tZ (20 μM) in 3-week-old wild-type plants. RNA was isolated at 0, 12, 24, 48, and 72 h after tZ treatment. Values are means ± SD; n = 3.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Overexpression of RLS1 Shows a Weak Accelerated Leaf Senescence Phenotype.
(A) RT–PCR analysis of RLS1 in wild-type and two independent representative RLS1 overexpression lines H426, H427. The rice ubiquitin (UBI-1) was used as a control.
(B) Leaf phenotypes of the same genotypes described in (A). F, flag leaf; 2, second leaf.
(C) Chlorophyll content in flag and second leaves of the same genotypes described in (A) was determined. Error bars indicate SD; n = 6; P < 0.05, by t-test.
(D) Membrane ion leakage. Error bars indicate SD; n = 6.
(E) Fv/Fm values. Error bars indicate SD; n = 6.
Molecular Plant 2012 5, DOI: ( /mp/ssr081)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions