Volume 1, Issue 5, Pages 732-750 (September 2008) The Receptor-Like Cytoplasmic Kinase (OsRLCK) Gene Family in Rice: Organization, Phylogenetic Relationship, and Expression during Development and Stress  Vij Shubha , Giri Jitender , Dansana Prasant Kumar , Kapoor Sanjay , Tyagi Akhilesh K.   Molecular Plant  Volume 1, Issue 5, Pages 732-750 (September 2008) DOI: 10.1093/mp/ssn047 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Domain Organization of the OsRLCK Protein Family. The SMART (http://smart.embl-heidelberg.de/) database was used to obtain the details of domain organization. Fourteen major types of domain organizations include RLCK (275), RLCKCK (10), RLCKDUF (4), RLCKLRR (23), RLCKWD40 (2), RLCKEGF/LECTIN (36), RLCKUBOX (15), RLCKUSP (6), RLCKUBQ (2), RLCKSPERM (2), RLCKJACALIN (1), RLCKECH (1), RLCKLYSM (1), and RLCKPPR (1). The asterisk on RLCKDUF, RLCKLRR, and RLCKWD40 indicates that the number and relative position of domains, namely DUF, LRR, and WD40, respectively, in these three categories of proteins could be variable. The filled circle on RLCKUBOX indicates that this domain is occasionally seen with some additional domains such as TPR and USP. The triangle mark on RLCKEGF/LECTIN is used to indicate that the domain organization for this class is only representative, since this particular class is much more diverse. Of the 36 RLCKEGF/LECTIN members, the actual organization is kinase+EGF (9), kinase+EGF+EGF (12), kinase+HDAC+Lectin (3), kinase+LECTIN (5), kinase+PAN+SLG+LECTIN (3), kinase+EGF+LECTIN (1), kinase+APPL+LECTIN (1), kinase+PAN+LECTIN (1), and kinase+PAN+EGF+LECTIN (1). Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Chromosomal Localization of the OsRLCK Gene Family Members. The genes are numbered in ascending order of their positions on the 12 rice chromosomes (refer to Table 1 for details). Filled circles indicate genes present in duplicated segments. Details are given in Supplemental Table 1. The association of genes falling within limits of abiotic stress-related QTLs available on the Gramene database (www.gramene.org) is shown. QTLs for the various traits are shown as follows; aluminum sensitivity (A1), cold tolerance (C1), deep root to shoot ratio (D1), drought susceptibility index (D2), elongation ability (E1), iron sensitivity (F1), KClO3 resistance (K1), potassium uptake (K2), leaf rolling (L1), leaf drying (L2), leaf rolling time (L3), lodging incidence (L4), sodium to potassium ratio (N1), sodium uptake (N2), sodium concentration (N3), osmotic adjustment capacity (O1), plant survival percentage under submergence (P1), phosphorus sensitivity (P2), penetrated root number (P3), penetrated to total root ratio (P4), penetrated root thickness (P5), relative phosphorus distribution between shoot and root, penetrated root length (P6) (R1), relative phosphorus concentration (R2), root penetration index (R3), relative growth rate (R4), root pulling force (R5), relative phosphorus uptake (R6), relative water content (R7), root weight (R8), relative phosphorus utilization efficiency (R9), rooting depth (R10), relative root length (R11), stomatal closure time (S1), stomatal resistance (S2), salt sensitivity (S3), total shoot elongation under submergence (T1), and ultraviolet-b resistance (U1). The differentially expressed genes during abiotic stress and falling in relevant QTL regions are also marked (*). Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phylogenetic Relationships amongst the Members of the OsRLCK Gene Family. The neighbor joining tree was generated using ClustalX for the 379 OsRLCK protein sequences and viewed using Treeview. Scale bar represents 0.1 amino acid substitutions per site. Based on bootstrap values, the proteins have been classified into 58 clades marked with red and blue colored lines. The six major clades (with ≥10 members each) are shaded in green. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Venn Diagram for Differentially Expressed OsRLCKs during Selected Reproductive Developmental Stages (Panicle and Seed) and under Abiotic Stress Conditions (Cold, Salt and Dehydration Stress). (A) Up-regulated OsRLCK s. (B) Down-regulated OsRLCK s. A gene is considered differentially expressed if it is up-or down-regulated at least two-fold, at P-value < 0.05, with respect to all vegetative stages (seedling, mature leaf, and root) in the case of reproductive development and 7-day-old seedling for the stress sample. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Expression Profiles of OsRLCK s under Reproductive Development Comprising Six Stages of Panicle (P1 (0–3 cm), P2 (3–5 cm), P3 (5–10 cm), P4 (10–15 cm), P5 (15–22 cm), and P6 (22–30 cm)) and Five Stages of Seed ((S1 (0–2 DAP), S2 (3–4 DAP), S3 (4–10 DAP), and S5 (11–20 DAP)) Development. A gene is considered differentially expressed during reproductive development if it is up-or down-regulated at least two-fold, at P-value < 0.05, with respect to all the vegetative controls, viz. 7-day-old seedling, mature leaf, and root. Clustering of the expression profile was done with log transformed average values taking mature leaf as base-line. The color scale at the bottom of the heat map is given in Log2 intensity value. The vertical color bars on the right represent genes that are differentially regulated more than two-fold in any one stage of panicle or seed development with reference to mature leaf. Based on the expression pattern, genes were grouped into five categories: A, up-regulated both in panicle and seed; B, up-regulated in panicle; C, up-regulated in seed; D, up-regulated in late panicle developmental stage (P6); E, down-regulated in both panicle and seed. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Expression Profiles of OsRLCK s under Three Abiotic Stress Conditions (CS: Cold Stress, DS: Dehydration Stress, SS: Salt Stress). A gene is considered differentially expressed under abiotic stress conditions if it is up-or down-regulated at least two-fold, at P-value < 0.05, with respect to 7-day-old seedling. Clustering of expression profile was done with log transformed average values taking seedling as base-line. The color scale is given in Log2 intensity value. The vertical color bars at the right side of the heat map represent genes differentially regulated more than two-fold in any one of the stress treatments with reference to seedling. Based on the expression pattern, genes were grouped into six categories: A, preferentially down-regulated in desiccation and/or salt stress; B, down-regulated in desiccation stress; C, up-regulated in salt stresses; D, up-regulated in desiccation stress; E, preferentially up-regulated in cold and/or desiccation stress; F, preferentially up-regulated in desiccation and/or salt stress. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Validation of Selected OsRLCK s Expression Profiles by Q–PCR. For real-time PCR analysis, the expression level of each gene was calculated in relation to its expression in unstressed 7-day-old seedlings and these values were normalized with the maximum average values obtained in the case of microarrays. White bars represent the expression from microarrays, while black bars represent the real-time PCR values. Error bar is shown. SDL, unstressed 7-day-old seedlings. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 8 Expression Categories of OsRLCK s Present in Segmentally and Tandemly Duplicated Regions of Rice Chromosomes. Based on microarray expression evidence, duplicated pairs were grouped into four different categories. Numbers in the stacked columns represent the number of duplicated pairs in the respective categories. For details of expression profiles, refer to Supplemental Figure 3. Molecular Plant 2008 1, 732-750DOI: (10.1093/mp/ssn047) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions