Introduction −1 2002 1982 1996 −1 1988 1997 1995 1988 1995 1995 2002 2000 1989 2000 2001 2001 2002 1982 2003 2002 2002 indica 2002 japonica japonica 2002 2003 indica japonica indica 2005 2005 indica 2005 Material and methods Plant materials for expression studies Oryza sativa 2002 2003 Plant culture and salinity treatments 1976 4 3 2 4 2 2 4 2 4 2 −2 −1 2002 2 1 w −1 w −1 Fig. 1 −1 2  Phenotypic characterization and gene expression studies + + −2 2 2 −1 2 −1 The plants were harvested 4 or 5 days after reaching PI. The main shoot was dissected for RNA extraction to obtain the growing point and crown tissue which was snap frozen. Approximately 12 plants were harvested per genotype per tank and tissue pooled to make each biological replicate for RNA extraction. Three biological replicates (one per array) were used for each treatment. Two plants from each tank were allowed to grow to maturity to verify that plants survived the imposed salinity stress. RNA extraction and processing for GeneChip analysis RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix, Inc., Santa Clara, CA) at the Core Instrumentation Facility at the University of California, Riverside by Barbara Walter. Total RNA was initially isolated from frozen shoot tissue using TRIzol Reagent. The RNA was purified using an RNeasy spin column (Qiagen, Chatsworth, CA) and an on-column DNase treatment. Eluted total RNAs were quantified with a portion of the recovered total RNA and adjusted to a final concentration of 1 μg/μl. All RNA samples were quality assessed prior to beginning target preparation/processing steps by running out a small amount of each sample (typically 25–250 ng/well) onto a RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) that was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (10 μg total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA) and poly(T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the Affymetrix GeneChip IVT Labeling Kit. Fifteen micrograms of the resulting biotin-tagged cRNA was fragmented to strands of 35–200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual). Subsequently, 10 μg of this fragmented target cRNA was hybridized at 45°C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 320) to probe sets present on an Affymetrix Rice Genome array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450 followed by scanning on a GeneChip Scanner 3000. Rice Genome array japonica indica http://www.ncbi.nlm.nih.gov/UniGene http://www.tigr.org/tdb/e2k1/osa1 Arabidopsis Statistical analysis of array data 2001 http://www.DChip.org 2 t P P http://www.ncbi.nlm.nih.gov/geo Expression validation by semi-quantitative RT-PCR japonica SKC1 2005 SKC1 Results 1 One confounding factor in experiment involving salinity stress is that salinity is known to delay PI in all genotypes. With this in mind we designed our experiment to harvest the control and salinity stressed tissue of each of the genotype at similar developmental stage i.e. 4–5 days after the plants reached PI. This was done because developmental equivalence when comparing control plants with stressed plants within each genotype is more relevant when the focus is on a specific sensitive stage such as PI. Shoot ion uptake of salt-tolerant and sensitive lines + 1 japonica + + + + + + + indicas + + japonica + + japonica − −1 −1 Table 1 Physiological responses of salt-sensitive and salt-tolerant genotypes Treatments + + − Tr M103 control 23.1 ± 1.1 872 ± 52.1 355 ± 30.4 12.1 ± 1.6 M103 salt 236 ± 16.5 881 ± 40.2 433 ± 32.5 8.9 ± 0.7 IR29 control 24 ± 3.0 799 ± 23.2 351 ± 24.3 10.8 ± 1.3 IR29 salt 279 ± 33.2 808 ± 49.5 525 ± 12.6 7.6 ± 0.3 Agami control 16.5 ± .96 772 ± 23 350 ± 30.5 7.0 ± 0.6 Agami salt 140 ± 20.1 688 ± 25.8 535 ± 12.1 4.2 ± 0.6 IR63731 control 25.7 ± 2.9 740 ± 30.5 279 ± 11 11.0 ± 0.8 IR63731 salt 166 ± 42.7 791 ± 33.2 545 ± 35.5 7.2 ± 0.8 + + − −1 −2 −1 1 japonica Expression responses of tolerant and sensitive genotypes to salinity stress japonica indica 1 t P japonica 2 japonica indica indica indica japonica 2 Fig. 2 A B C japonica D indica P  Table 2 P Putative function Probe set Rice locus P IR29 M103 IR63731 Agami WSI76 Os.2677.1.S1_at Os07g48830 0.002 0.01 0.018 0.038 LTI30 Os.12633.1.S1_s_at Os11g26790 0.001 0.009 0.002 0.0005 Myb-like DNA-binding domain Os.10333.1.S1_at Os07g02800 0.0001 0.001 0.001 0.001 RNase S-like protein (drought induced) Os.12922.1.S1_at Os09g36700 0.002 0.0004 0.00006 0.004 MtN3 Os.16044.1.S1_at Os02g30910 0.036 0.026 0.0003 0.006 Low affinity nitrate transporter Os.32686.1.S1_at Os01g65110 0.0003 0.0009 0.0168 0.007 Putative Myb-like DNA-binding Protein Os.25453.1.A1_at Os03g55590 0.0002 0.0027 0.0036 0.015 Bowman–Birk serine protease inhibitor Os.7612.1.S1_at Os03g60840 0.014 0.012 0.024 0.008 NTP2 Os.45923.1.S1_at Os01g37590 0.0005 0.0003 0.0043 0.0013 O OsAffx.4277.1.S1_at Os05g08750 0.007 0.023 0.012 0.006 Arabidopsis japonica indica 2 japonicas indica GPL4 indica RD22 NR1 RD22 1993 http://www.tigr.org 3 2002 2004 indica 3 Saltol Saltol 2004 2002 Fig. 3 A B  Genes involved in ion homeostasis + + + 3 P CLC-d P + KT HAK KUP + 1997 HAK4 HAK5 KAT3 KUP1 P Table 3 Genes from selected categories which were differentially expressed under saline conditions in one or more rice genotypes Putative function Probe set Locus M103 IR29 IR63731 Agami Ion homeostasis + Os.2358.1.S1_at Os08g36340 1.2 0.63 – – CLC-d chloride channel Os.27207.1.S1_at Os12g25200 4.16 2.88 0.78 – Cation channel protein (KAT3) OsAffx.21536.1.S1 Os01g52070 −1.05 – – – + 2+ Os.20354.1.S1_at Os03g45370 −1.11 – – – + Os.27454.1.S1_x Os07g01220 −1.54 – – – + Os.50590.2.A1_at Os02g07830 – 1.0 – – + Os.6037.2.S1_x_at Os09g27580 – 2.51 – – Flowering and inflorescence Centroradialis protein (CEN) Os.57548.1.S1_at At2g27550 1.55 1.44 – – Zinc finger protein (ID1) Os.4766.1.S1_at  1.47 0.62 – – Constan-like protein (CO6) Os.7920.1.S1_at Os06g44450 1.39 1.0 0.68  CCT motif Os.16422.1.S1_s_at Os08g15050 1.04 – – – CLAVATA1 receptor kinase Os.7123.2.S1_x_at  0.82 1.05 – – AP1-like MADS-box protein Os.2348.1.S1_a_at  −0.91 −1.09 – – Gigantea-like protein Os.7987.1.S1_at Os01g08700 −1.04 – – −1.74 Floral homeotic protein (AP1) Os.12750.2.S1_s_at At1g69120 −1.12 – – – Flowering locus T OsAffx.15765.1.S1 Os06g35940 −1.13 1.7 – – Homeodomain (KNAT7) Os.4164.2.S1_a_at At1g62990 −1.66 – – – Sex determination tasselseed 2 Os.15281.1.S1_at  – 3.93 – – No apical meristem (NAM) Os.37548.1.S1_at  – 1.81 – – Auxin CYP83B1 Os.11110.1.S1_at At4g31500 −1.71 – – – Auxin-repressed protein ARP1 Os.12735.1.S1_at  1.21 – – – Auxin-responsive protein Os.20151.1.S1_at At3g25290 – 1.27 – – Auxin transporter PIN1 Os.37330.1.S1_at  −0.94 – – −1.1 Axi1 Os.52961.1.S1_at  −1.0 – – – Phototropic-responsive NPH3 Os.56849.1.S1_at At1g03010 1.03 – – – NPK1-related protein kinase Os.5940.1.S1_at  – 1.46 – – Auxin response factor 10 Os.8374.1.S1_at Os04g43910 −1.17 – – – Anthocyanin pathway Chalcone, stilbene synthases Os.11154.1.S1_a Os03g06700 1.11 1.46 0.92 1.3 Phenylalanine ammonia-lyase Os.25687.1.S1_x Os02g41680 – 1.25 – – Isoflavone reductase Os.32454.1.S1_at At4g39230 −1.96 – – – 4-coumaroyl-CoA synthase 3 Os.4377.1.S1_at At1g65060 −1.4 – – – Flavonoid 3′-hydroxylase  Os.46551.1.S1_at At5g07990 – −1.09 – – Dihydroflavonol 4-reductase Os.48545.1.S1_at Os04g53810 – 1.94 – – Ferulate-5-hydroxylase Os.9727.1.S1_at At4g36220 −1.64 – – – Chalcone isomerase (CHI) Os.9929.1.S1_at At3g55120 −1.31 – – – 5-hydroxyferulic acid OsAffx.13783.1.S1 At5g54160 – 1.02 – – 2 2 P + P P indica P Saltol 2002 SKC1 2005 2005 4 Fig. 4 SKC1 indica SCK1  Differential expression of auxin associated genes in rice under salinity stress P 2000 axi1 NPK1 NPH3 NPH3 2004 PIN1 3 Effect of salinity stress on floral transition and development in rice 1999 CEN indeterminant1 ID1 P P FT gigantea gigantea-like japonica gigantea FT TFL1 EMF2 3 Discussion indicas japonicas. + + WSI76 LTI30 WSI76 1994 2005 NRT1 Mesembryanthemum crystallinum 2003 NRT2 2 Ion homeostasis in rice under salinity stress + + Arabidopsis HKT1 2006 + + 2005 1 + + + SKC1 2005 SKC1 + 2004 SKC1 HKT + SKC1 2005 SKC1 SKC1 + + 1 SKC1 2005 indica SKC1 SKC1 indicas 4 SKC1 indica SKC1 Auxin and response of rice to salinity stress IAOx 2001 axi1 1992 1993 1995 1997 − 1973 1974 1998 1973 − Petroselinum sativum 1998 − − − 1 CLC-d P P NPK1 MAPKK NPK1 1998 2000 sos2-1 Arabidopsis 2005 Salinity stress and floral transition 2002 terminal flower 1 TFL1 centroradialis CEN TFL1/CEN 2002 TFL1/CEN TFL1 CEN 1996 FT FT TFL1 FT FT FT FT 2005 FT FT gigantea GI gigantea OsGI 2003 gigantea-like 3 indica 2002 Commonalties in the salinity stress response of IR29 at early vegetative versus reproductive stage 2005 japonica SALT Saltol SALT 2005 Saltol salT 1990 1997 OPR2 JIP 2005 + 2007 salT + 2001 + 2004 STZ/ZAT10 STZ/ZAT10 COR/RD 2002 STZ/ZAT10 Electronic supplementary materials Below are the links to the electronic supplementary materials.
 ESM 1 (XLS 64 kb) ESM 2 (XLS 74 kb) ESM 3 (XLS 21 kb) ESM 4 (XLS 21 kb) ESM 5 (XLS 111 kb) ESM 6 (XLS 23 kb) ESM 7 (XLS 21 kb) ESM 8 (XLS 18 kb) ESM 9 (XLS 126 kb) ESM 10 (XLS 32 kb) ESM 11 (XLS 128 kb) ESM 12 (XLS 29 kb) ESM 13 (XLS 16 kb)