Molecular genetic analysis of cold–regulated gene transcription

Chilling and freezing temperatures adversely affect the productivity and quality of crops. Hence improving the cold hardiness of crop plants is an important goal in agriculture, which demands a clear understanding of cold stress signal perception and transduction. Pharmacological and biochemical evidence shows that membrane rigidification followed by cytoskeleton rearrangement, Ca 2+ influx and Ca 2+ –dependent phosphorylation are involved in cold stress signal transduction. Cold–responsive genes are regulated through C–repeat/dehydration–responsive elements (CRT/DRE) and abscisic acid (ABA)–responsive element cis elements by transacting factors C–repeat binding factors/dehydration–responsive element binding proteins (CBFs/DREBs) and basic leucine zippers (bZIPs) (SGBF1), respectively. We have carried out a forward genetic analysis using chemically mutagenized Arabidopsis plants expressing cold–responsive RD29A promoter–driven luciferase to dissect cold signal transduction. We have isolated the fiery1 ( fry1 ) mutant and cloned the FRY1 gene, which encodes an inositol polyphosphate 1–phosphatase. The fry1 plants showed enhanced induction of stress genes in response to cold, ABA, salt and dehydration due to higher accumulation of the second messenger, inositol (1,4,5)– triphosphate (IP 3 ). Thus our study provides genetic evidence suggesting that cold signal is transduced through changes in IP 3 levels. We have also identified the hos1 mutation, which showed super induction of cold–responsive genes and their transcriptional activators. Molecular cloning and characterization revealed that HOS1 encodes a ring finger protein, which has been implicated as an E3 ubiquitin conjugating enzyme. HOS1 is present in the cytoplasm at normal growth temperatures but accumulates in the nucleus upon cold stress. HOS1 appears to regulate temperature sensing by the cell as cold–responsive gene expression occurs in the hos1 mutant at relatively warm temperatures. Thus HOS1 is a negative regulator, which may be functionally linked to cellular thermosensors to modulate cold–responsive gene transcription.