Liming Xiong

Cell Signaling during Cold, Drought, and Salt Stress

Low temperature, drought, and high salinity are common stress conditions that adversely affect plant growth and crop production. The cellular and molecular responses of plants to environmental stress have been studied intensively ([Thomashow, 1999][1]; [Hasegawa et al., 2000][2]). Understanding the

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.

Genetic Analysis of Salt Tolerance in Arabidopsis: Evidence for a Critical Role of Potassium Nutrition

A large genetic screen for sos (for salt overly sensitive) mutants was performed in an attempt to isolate mutations in any gene with an sos phenotype. Our search yielded 28 new alleles of sos1, nine mutant alleles of a newly identified locus, SOS2, and one allele of a third salt tolerance locus, SOS3. The sos2 mutations, which are recessive, were mapped to the lower arm of chromosome V, ~2.3 centimorgans away from the marker PHYC. Growth measurements demonstrated that sos2 mutants are specifically hypersensitive to inhibition by Na+ or Li+ and not hypersensitive to general osmotic stresses. Interestingly, the SOS2 locus is also necessary for K+ nutrition because sos2 mutants were unable to grow on a culture medium with a low level of K+. The expression of several salt-inducible genes was superinduced in sos2 plants. The salt tolerance of sos1, sos2, and sos3 mutants correlated with their K+ tissue content but not their Na+ tissue content. Double mutant analysis indicated that the SOS genes function in the same pathway. Based on these results, a genetic model for salt tolerance mechanisms in Arabidopsis is presented in which SOS1, SOS2, and SOS3 are postulated to encode regulatory components controlling plant K+ nutrition that in turn is essential for salt tolerance.

The Arabidopsis LOS5/ABA3 Locus Encodes a Molybdenum Cofactor Sulfurase and Modulates Cold Stress– and Osmotic Stress–Responsive Gene Expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.

Regulation of abscisic acid biosynthesis.

Plant growth and development are regulated by internal signals and by external environmental conditions. One important regulator that coordinates growth and development with responses to the environment is the sesquiterpenoid hormone abscisic acid (ABA). ABA plays important roles in many cellular

Modulation of Abscisic Acid Signal Transduction and Biosynthesis by an Sm-like Protein in Arabidopsis

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.

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.

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.

Identification of Drought Tolerance Determinants by Genetic Analysis of Root Response to Drought Stress and Abscisic Acid

Drought stress is a common adverse environmental condition that seriously affects crop productivity worldwide. Due to the complexity of drought as a stress signal, deciphering drought tolerance mechanisms has remained a major challenge to plant biologists. To develop new approaches to study plant drought tolerance, we searched for phenotypes conferred by drought stress and identified the inhibition of lateral root development by drought stress as an adaptive response to the stress. This drought response is partly mediated by the phytohormone abscisic acid. Genetic screens using Arabidopsis (Arabidopsis thaliana) were devised, and drought inhibition of lateral root growth (dig) mutants with altered responses to drought or abscisic acid in lateral root development were isolated. Characterization of these dig mutants revealed that they also exhibit altered drought stress tolerance, indicating that this root response to drought stress is intimately linked to drought adaptation of the entire plant and can be used as a trait to access the elusive drought tolerance machinery. Our study also revealed that multiple mechanisms coexist and together contribute to whole-plant drought tolerance.