Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of arabidopsis encoding highly conserved hydrophobic proteins

Abstract

The capability of most higher plants to tolerate environmental conditions strongly depends on their developmental stage. In addition, environmental factors have pleiotropic effects on many developmental processes. The interaction between plant development and environmental conditions implies that some genes must be regulated by both environmental factors and developmental cues. To understand their developmental regulation and obtain possible clues on their functions, we have isolated genomic clones for RCI2A and RCI2B, two genes from Arabidopsis ecotype Columbia (Col), whose expression is induced in response to low temperature, dehydration, salt stress, and abscisic acid. The promoters of RCI2A and RCI2B were fused to the uidA (GUS)-coding sequence and the resulting constructs used to transform Arabidopsis. GUS activity was analyzed in transgenic plants during development under both stressed and unstressed conditions. Transgenic plants with either the RCI2A or RCI2B promoter showed strong GUS expression during the first stages of seed development and germination, in vascular bundles, pollen, and most interestingly in guard cells. When transgenic plants were exposed to low temperature, dehydration, salt stress, or abscisic acid, reporter gene expression was induced in most tissues. These results indicate that RCI2A and RCI2B are regulated at transcriptional level during plant development and in response to different environmental stimuli and treatments. The potential role of RCI2A and RCI2B in plant development and stress response is discussed.

Figure 1

Molecular characterization of RCI2A and RCI2B genes. A, Organization of RCI2A and RCI2B in the 4.5-kb EcoRI genomic fragment. Exons are shown as white boxes and introns as black boxes. Transcriptional orientation is indicated by arrows. B, Sequence comparison of RCI2A and RCI2B promoter regions. Transcription start sites are indicated by +1 and underlined. The initiation ATGs codons, and putative TATA boxes are double underlined. G-Box (CACGTG) and related sequences are shown in gray boxes. Black boxes highlight LTRE/DRE/C-repeat (CCGAC) and related sequences. MYB-like sequences (TAACCA) are indicated as open boxes. The MYC-like sequence (CACATG) is shaded. The pollen-specific regulatory element AAATGA is underlined. Identical nucleotides are indicated by a line. Points represent gaps inserted into the sequences for optimal alignment.

Figure 2

Sequence similarity between RCI2 and related proteins. Alignment of amino acid sequences of RCI2 and similar proteins from Hordeum vulgare (BLT101; accession no. Z25537), L. elongatum (ESI3; accession no. U00966), Saccharomyces cerevisiae (PMP3; accession no. X91499), Synechocystis sp. (SSR1169; accession no. P74805), E. coli (YQAE; accession no. P77240), and Caenorhabditis elegans (T23F23; accession no. Q22700). Identical amino acid residues to RCI2A sequence are indicated by asterisks. Dashes represent gaps inserted into the sequences for optimal alignment. Gray boxes highlight the putative transmembrane domains. The number of residues of each sequence is indicated in the right side.

Figure 3

GUS activity in transgenic seedlings of Arabidopsis carrying RCI2A- and RCI2B-GUS fusions, exposed to different treatments. Ten-day-old seedlings were used as controls (C), exposed to 4°C for 24 h (COLD), dehydrated till losing 50% of fresh weight (DH), treated with 100 μm ABA (ABA), and exposed to 250 mm NaCl for 12 h (NaCl). Data are expressed as means (n = 15; five plants of tree independent transformant lines). Bars indicate se. In all cases, values obtained from treated and control plants were significantly different (P < 0.05) as determined by Student's t test.

Figure 4

Expression of RCI2A and RCI2B during seed germination. A, Histochemical localization of GUS activity in germinating transgenic seeds containing the RCI2A-GUS fusion. Seeds were placed on filter paper soaked with distilled water and stained for GUS activity 0, 1, 2, 3, and 4 d after germination. Identical results were obtained with RCI2B-GUS germinating seeds. B, RNA-blot analysis of RCI2A and RCI2B genes in germinating seeds. Total RNA (10 μg) obtained from matured (0) and 1 to 6 d (1d–6d) germinating seeds was loaded on each line. The specific probes for RCI2A and RCI2B are described in Capel et al. (1997). The ethidium bromide-stained gel is shown at bottom as a control of equal RNA loading.

Figure 5

Histochemical localization of GUS activity in transgenic Arabidopsis seedlings containing RCI2A- and RCI2B-GUS fusions grown under control conditions or exposed to 4°C for 24 h. A through C, Seven-day-old RCI2A-GUS seedlings grown under control conditions. A, Whole seedling; B, cotyledon. The close up shows GUS staining in guard cells. C, Hypocotyl; D through F, 7-d-old RCI2A-GUS seedlings exposed to 4°C for 24 h; D, whole seedling; E, cotyledon; F, hypocotyl; G through I, 7-d-old RCI2B-GUS seedlings grown under control conditions; G, whole seedling; H, cotyledon. The close up shows strong GUS staining in guard cells. I, Hypocotyl; J through L, 7-d-old RCI2B-GUS seedlings exposed to 4°C for 24 h. J, Whole seedling; K, cotyledon; L, hypocotyl.

Figure 6

Histochemical localization of GUS activity in adult transgenic Arabidopsis plants containing RCI2A- and RCI2B-GUS fusions grown under control conditions or exposed to 4°C for 24 h. A through I, RCI2A-GUS plants grown under control conditions. A, Root; B, root primordia; C, cross-section of a stem. Arrows indicate GUS staining in protoxylem cells. The inside shows a general view of the section. D, Leaf. The close up shows GUS staining in guard cells. E, Cross-section of a leaf; F, flower; G, pollen grains; H, inmature silique. The close up shows GUS staining in ovules. I, Mature silique. The close up shows mature seeds. J through N, RCI2A-GUS plants exposed to 4°C for 24 h. J, Root; K, root primordia; L, cross-section of a stem. The inside shows a general view of the section. M, Leaf; N, cross-section of a leaf. Identical results were obtained with RCI2B-GUS plants.