Becky Stevenson

Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways.

To dissect genetically the complex network of osmotic and cold stress signaling, we constructed lines of Arabidopsis plants displaying bioluminescence in response to low temperature, drought, salinity, and the phytohormone abscisic acid (ABA). This was achieved by introducing into Arabidopsis plants a chimeric gene construct consisting of the firefly luciferase coding sequence (LUC) under the control of the stress-responsive RD29A promoter. LUC activity in the transgenic plants, as assessed by using in vivo luminescence imaging, faithfully reports the expression of the endogenous RD29A gene. A large number of cos (for constitutive expression of osmotically responsive genes), los (for low expression of osmotically responsive genes), and hos (for high expression of osmotically responsive genes) mutants were identified by using a high-throughput luminescence imaging system. The los and hos mutants were grouped into 14 classes according to defects in their responses to one or a combination of stress and ABA signals. Based on the classes of mutants recovered, we propose a model for stress signaling in higher plants. Contrary to the current belief that ABA-dependent and ABA-independent stress signaling pathways act in a parallel manner, our data reveal that these pathways cross-talk and converge to activate stress gene expression.

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.

LOS2, a genetic locus required for cold-responsive gene transcription encodes a bi-functional enolase

The Arabidopsis mutation, los2, impairs cold‐responsive gene transcription, acquired freezing tolerance and plant resistance to chilling under certain conditions. LOS2 was isolated through positional cloning and shown to encode an enolase in the glycolytic pathway. In animal cells, enolase has also been known to function as a transcription factor that represses the expression of c‐myc by binding to the c‐myc gene promoter. LOS2 fused to green fluorescent protein is targeted to the nucleus as well as to the cytoplasm. LOS2/enolase protein can bind to the cis‐element of the human c‐myc gene promoter and to the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor from Arabidopsis. STZ/ZAT10 expression is induced rapidly and transiently by cold in the wild type, and this induction is stronger and more sustained in the los2 mutant. Furthermore, the expression of a RD29A‐LUC reporter gene is repressed significantly by STZ/ZAT10 in transient expression assays in Arabidopsis leaves. Our results demonstrate that cold‐responsive gene transcription in plants is controlled by a bi‐functional enolase.

Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress

C2H2‐zinc finger proteins that contain the EAR repressor domain are thought to play a key role in modulating the defense response of plants to abiotic stress. Constitutive expression of the C2H2‐EAR zinc finger protein Zat10 in Arabidopsis was found to elevate the expression of reactive oxygen‐defense transcripts and to enhance the tolerance of plants to salinity, heat and osmotic stress. Surprisingly, knockout and RNAi mutants of Zat10 were also more tolerant to osmotic and salinity stress. Our results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.

HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19°C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10°C or higher. Compared with the wild type, hos1-1 plants are less cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.

A DEAD Box RNA Helicase Is Essential for mRNA Export and Important for Development and Stress Responses in Arabidopsis

An Arabidopsis thaliana mutant, cryophyte, was isolated and found to have an enhanced cold stress-induction of the master regulator of cold tolerance, C-repeat binding factor 2 (CBF2), and its downstream target genes. The mutant is more tolerant to chilling and freezing stresses but is more sensitive to heat stress. Under warm but not cold growth temperatures, the mutant has a reduced stature and flowers earlier. Under long day conditions, flowering of the mutant is insensitive to vernalization. The mutant is also hypersensitive to the phytohormone abscisic acid. The mutation was found in a DEAD box RNA helicase gene that is identical to the previously identified low expression of osmotically responsive genes 4 (LOS4) locus, which was defined by the los4-1 mutation that reduces cold regulation of CBFs and their target genes and renders Arabidopsis plants chilling sensitive. We show evidence suggesting that the CRYOPHYTE/LOS4 protein may be enriched in the nuclear rim. In situ poly(A) hybridization indicates that the export of poly(A)+ RNAs is blocked in the cryophyte/los4-2 mutant at warm or high temperatures but not at low temperatures, whereas the los4-1 mutation weakens mRNA export at both low and warm temperatures. These results demonstrate an important role of the CRYOPHYTE/LOS4 RNA helicase in mRNA export, plant development, and stress responses.

RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance.

Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis.

Low temperature, drought, and high salinity induce the expression of many plant genes. To understand the mechanisms for the transcriptional activation of these genes, we conducted a reporter gene-aided genetic screen in Arabidopsis. Seven allelic mutations in the FIERY2 (FRY2) locus result in significant increases in the expression of stress-responsive genes with the DRE/CRT (drought-responsive/C-repeat) cis element but non-DRE/CRT type stress-responsive genes were less affected. The specific regulation of DRE/CRT class of genes by FRY2 appears to be caused by repression of stress induction of the upstream CBF/DREB transcription factor genes. fry2 mutants show increased tolerance to salt stress and to abscisic acid during seed germination but are more sensitive to freezing damage at the seedling stage. FRY2/CPL1 encodes a novel transcriptional repressor harboring two double-stranded RNA-binding domains and a region homologous to the catalytic domain of RNA polymerase II C-terminal domain phosphatases found in yeast and in animals that regulate gene transcription. These data indicate that FRY2 is an important negative regulator of stress gene transcription and suggest that structured RNA may regulate hormone and stress responses in plants as it does in animals.

PARTIAL SEQUENCING AND MAPPING OF CLONES FROM TWO MAIZE CDNA LIBRARIES

As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, ‘single-pass’ sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total of 313 clones from the two libraries were submitted to ‘single-pass’ sequencing from the presumed 5′ end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.

Disruption of Arabidopsis CHY1 Reveals an Important Role of Metabolic Status in Plant Cold Stress Signaling

To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter (CBF3-LUC). One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chy1 mutant. chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chy1 mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chy1 plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wild-type but not in chy1. Constitutive CBF3 expression partially rescues the sensitivity of chy1-10 plants to dark treatment in the cold. The chy1 mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal beta-hydroxyisobutyryl (HIBYL)-CoA hydrolase needed for valine catabolism and fatty acid beta-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.