Paper 1 Flashcards
discussion
what were the questions of this paper?
What are core regulators of lignification and suberization at the rice endodermis?
How do lignification and suberization at the endodermis interact?
How do endodermal lignification and suberization interplay with stress signals?
what were the main conclusions of this paper?
Four MYB transcription factor genes, OsMYB39a, OsMYB41, OsMYB92a, and OsMYB92b, are identified as core regulators of suberization and nonlocalized lignification at the rice endodermis, acting downstream of OsMYB36a/b/c, OsCIF-OsSGN3, and stress-inducible signaling pathways.
Knockout of all 4 genes resulted in a complete lack of endodermal suberin lamellae (SL) and nearly no lignin deposition between the Casparian strip and the cortex-facing lignified band at cell corners under normal and stress conditions.
Stress stimuli strongly accelerated nonlocalized lignification and SL formation at the endodermis of the wild type.
There is a tight link between SL formation and gapless lignification along the anticlinal cell walls at the endodermis.
Endodermis-specific overexpression of OsMYB92a induced an early formation of SL in the root tip and reduced Na accumulation in shoots, thus significantly enhancing rice tolerance to salinity stress.
Figure 1
regulation
A to D: Shows the expression of OsMYB39a/41/92a/92b in the Osmyb36abc triple mutant. RNA was extracted from the roots of 21-day-old wild-type (WT) and Osmyb36abc-1 seedlings. The expression of the 4 OsMYB genes was analyzed using RT-qPCR. Histone H3 was used as an internal standard. Data are presented as means ± SD of 3 biological replicates.
E: Is a schematic diagram of the reporter and effector constructs used in the dual luciferase assay system.
F to I: Depicts the activation of the promoters of OsMYB39a (F), OsMYB41 (G), OsMYB92a (H), and OsMYB92b (I) by OsMYB36s in rice protoplasts. The LUC/REN ratio relative to CK (a basal signal without OsMYB36s) is shown. Data are means ± SD of 3 biological replicates.
J: Shows OsMYB36 proteins binding to the promoter of OsMYB92a in yeast.
K: Demonstrates EMSA (electrophoretic mobility shift assay) results showing the binding of recombinant His-OsMYB36s-N to the MYB-binding motif of the OsMYB92a promoter.
figure 2
expression pattern
A: Shows the relative expression of OsMYB39a/41/92a/92b in various tissues of wild-type (WT) rice. Different tissues from 90-day-old WT rice were sampled for RNA extraction and expression analysis.
B: Depicts the spatial expression of OsMYB39a/41/92a/92b in the roots. RNA was extracted from different segments of the roots (0 to 1, 1 to 2, 2 to 3, and 3 to 4 cm from the root apex) of 5-day-old seedlings.
C to F: Shows the expression of OsMYB39a (C), OsMYB41 (D), OsMYB92a (E), and OsMYB92b (F) in response to ABA and Cd stresses. Rice seedlings were exposed to a 0.5 mM CaCl2 solution containing 0, 10 µM ABA, or 10 µM CdSO4 for 12 hours. Expression of 4 OsMYB genes was analyzed by RT-qPCR.
G to P: Illustrates the cell-specific GUS expression of ProOsMYB92a:GUS transgenic lines in the primary roots.
G to J: Shows GUS expression detected in whole roots except for the root tip. H to J are magnified images of the black boxed area in (G).
K to P: Displays transverse sections of the roots (10, 20, and 30 mm from the root apex) of 5-day-old seedlings, revealing the expression of GUS in the endodermis and stele. N to P are magnified images.
figure 3
presents observations of lignin deposition in the wild-type (WT) and OsMYBs-knockout lines.
Lignin staining was performed on roots at 30 and 40 mm from the apex of the WT (A to A2, H to H2), Osmyb92a-1 (B to B2, I to I2), Osmyb92a-2 (C to C2, J to J2), Osmyb41/92a (D to D2, K to K2), Osmyb41/92a/92b (E to E2, L to L2), Osmyb39a/41/92a/92b-1 (F to F2, M to M2), and Osmyb39a/41/92a/92b-2 (G to N2).
O, Q: Show the percentage of pericycle-facing broad bands at the endodermis.
P, R: Show the percentage of cortex-facing broad bands at the endodermis.
A1 to N1: Are magnified images of the yellow boxed area in (A to N), respectively.
A2 to N2: Are magnified images of the yellow boxed area in (A1 to N1), respectively. A1 to N1 and A2 to N2 are 3D images constructed from z-stacks.
magenta and blue signals to indicate lignin and cellulose, respectively. Arrows of different colors indicate specific structures: yellow arrows indicate the Casparian strip (CS), red arrows indicate the lignified band at the cortex-facing corners, and white arrows indicate the lignified band at the pericycle-facing corners.
figure 4
suberin deposition at the endodermis in the wild type (WT) and OsMYBs-knockout lines under normal and stress conditions.
Suberin was stained by Fluorol Yellow (FY) 088 in the roots (30 mm from the apex) of the WT (A, H, O, V), Osmyb92a-1 (B, I, P, W), Osmyb92a-2 (C, J, Q, X), Osmyb41/92a (D, K, R, Y), Osmyb41/92a/92b (E, L, S, Z), Osmyb39a/41/92a/92b-1 (F, M, T, AA), and Osmyb39a/41/92a/92b-2 (G, N, U, AB).
Five-day-old seedlings of all lines were exposed to a 0.5 mM CaCl2 solution containing 0 (A to G), 10 µM ABA (H to N), 100 mM NaCl (O to U), or 10 µM CdSO4 (V to AB) for 24 hours. Yellow shows the signal of suberin.
AC to AF: Show the percentage of suberized cells at the endodermis 30 mm from the root apex under normal (AC), 10 µM ABA (AD), 100 mM NaCl (AE), or 10 µM CdSO4 (AF) treatments.
AG to AJ: Present the mean pixel intensity of suberin at the endodermis 30 mm from the root apex under normal (AG), 10 µM ABA (AH), 100 mM NaCl (AI), or 10 µM CdSO4 (AJ) treatments.
AK: Shows aliphatic suberin monomer compounds in 5-day-old roots of the WT and Osmyb39a/41/92a/92b mutants. Each suberin component is designated by carbon chain length and labeled by chemical class along the x-axis.
AL: Shows the total amount of suberin monomer in roots.
figure 5
lignin and suberin deposition, as well as propidium iodide (PI) penetration in the wild-type (WT) and OsMYB92a-overexpressing lines.
A to C: Lignin staining was performed in the roots (5 mm from the apex) of the WT (A to A2), ProOsCASP1:OsMYB92a-1 (B to B2), and ProOsCASP1:OsMYB92a-2 (C to C2).
D to F: Staining for suberin (yellow) was performed to observe suberin deposition at the endodermis from the root apex (5 mm) of WT (D), ProOsCASP1:OsMYB92a-1 (E), and ProOsCASP1:OsMYB92a-2 (F).
G to L: PI penetration test in the roots of WT (G-G1, J), ProOsCASP1:OsMYB92a-1 (H to H1, K), and ProOsCASP1:OsMYB92a-2 (I to I1, L). Cross-sections are observed at 5 mm (G to I) and 10 mm (J to L) from the root apex.
M: Width of the lignified band at the endodermis.
N: Thickness of the Casparian strip (CS) at the endodermis.
O: Percentage of broad lignified bands at the endodermis.
P: Percentage of suberized cells at the endodermis.
figure 6
regulation of nonlocalized lignification- and suberin (SL)-associated genes by OsMYB39a/41/92a/92b.
A: Venn diagrams of downregulated genes by Osmyb39a/41/92a/92b and upregulated genes by Cd and ABA treatments.
B: Cluster analysis of nonlocalized lignification- and SL-associated genes regulated by OsMYB39a/41/92a/92b.
C: Schematic diagram of the reporter and effector constructs used in the dual luciferase assay system.
D, E: Activation of the promoters of OsCYP86A1 (D) and OsGELP82 (E) by OsMYB92a in rice protoplasts.
F, G: OsMYB92a binding to the promoters of OsCYP86A1 (F) and OsGELP82 (G) in yeast.
H to J: EMSA showing binding of recombinant His-OsMYB92a-N to the MYB-binding motif of target promoters.
K to O: Observation of suberin deposition at the endodermis in the Osgelp43/81/82/111 and Oscyp86a1 mutants.
P, R: Percentage of suberized cells at the endodermis (P) or exodermis (R).
Q, S: Mean pixel intensity of suberin at the endodermis (Q) or exodermis (S).
figure7
wild-type (WT) rice, Osmyb39a/41/92a/92b mutants, and OsMYB92a-overexpressing lines.
A: Shows the concentration of K, P, Mg, and Ca in the shoots.
B: Shows the concentration of Cd and As in the shoots.
C: Shows the concentration of Fe, Mn, Na, Zn, Rb, Ge, Cu, and Sr in the shoots.
D: Shows the growth performance of rice seedlings under control conditions (0 mM NaCl).
E: Shows the growth performance of rice seedlings under salt conditions (75 mM NaCl).
F: Shows the dry weight of the shoots.
G: Shows the dry weight of the roots.
H: Shows the tissue Na concentration in WT rice, Osmyb39a/41/92a/92b mutants, and OsMYB92a-overexpressing lines under salt conditions.
describe figure 8
the localization of the silicon (Si) transporter Lsi1 in wild-type (WT) rice, Osmyb39a/41/92a/92b mutants, and OsMYB92a-overexpressing lines.
A to E: Immunostaining with an anti-Lsi1 antibody was performed in the roots at 20 mm from the apex of WT (A and B), Osmyb39a/41/92a/92b-1 (C), ProOsCASP1:OsMYB92a-1 (D), and ProOsCASP1:OsMYB92a-2 (E). B is a magnified image of the yellow boxed area in (A). Green shows the signal of Lsi1, and blue shows cellulose.
F: Percentage of cells expressing Lsi1 at the endodermis.
G: Mean pixel intensity of Lsi1 at the endodermal cells.
key points Four MYB Transcription Factors Identified
The study identifies OsMYB39a, OsMYB41, OsMYB92a, and OsMYB92b as key regulators of suberization and nonlocalized lignification in rice root endodermis.
key points Regulation of Suberization and Lignification:
These MYB factors act downstream of OsMYB36a/b/c, OsCIF-OsSGN3, and stress-inducible signaling pathways.
key points Impact of Gene Knockout
Knocking out all four MYB genes leads to a complete absence of endodermal suberin lamellae and significantly reduces lignin deposition.
key points stress response
Stressful conditions like ABA, NaCl, and Cd treatments accelerate nonlocalized lignification and suberin formation in wild-type rice.
key points Link Between Lignification and Suberization:
The research highlights a strong connection between suberin lamellae formation and complete lignification along the endodermal cell walls.
key points OsMYB92a Overexpression and Salinity Tolerance:
Overexpressing OsMYB92a in the endodermis promotes early suberin lamellae formation, reduces sodium accumulation, and enhances salinity tolerance in rice.
key points Regulation of Downstream Genes
The MYB transcription factors regulate genes involved in lignin and suberin biosynthesis and polymerization, including OsCYP86A1 and OsGELP genes.
key points Role in Ion Homeostasis:
The study suggests that endodermal suberization and lignification, regulated by these MYB factors, play a critical role in ion homeostasis and acclimation to environmental stress, particularly salinity.
key points Potential Applications:
The findings suggest potential applications of OsMYB92a in breeding rice lines with increased tolerance to abiotic stresses.
key points Conserved Pathway:
The components of the pathway responsible for endodermal suberization are conserved between Arabidopsis and rice.
