cold stress ͉ RING finger protein I n response to low temperatures, numerous genes are induced in plants (1). Three transcription factors known as C-repeat (CRT)-binding factors (CBFs) or dehydration-responsive element (DRE)-binding protein can bind to CRT͞DRE cis elements in the promoters and activate transcription of many of the cold-responsive genes (2-4). The CBF genes are transiently induced by low temperature, and this induction precedes that of the downstream cold-responsive genes (4-6). ICE1 is an upstream constitutively expressed transcription factor that controls the cold induction of CBF3 (7).Previously, a genetic screen using Arabidopsis plants that express the firefly luciferase reporter gene driven by the CRT͞ DRE element-containing RD29A promoter led to the identification of HOS1 (high expression of osmotically responsive gene 1), a negative regulator of the CBF regulon (8). CBFs and their downstream genes show enhanced cold induction in loss-offunction hos1 mutant plants (8). HOS1 was predicted to encode a 915-aa protein containing a short motif near the N terminus that is similar to the RING finger domain found in a group of animal proteins known as inhibitor of apoptosis (9). Recently, several plant proteins containing RING finger domain have been shown to function as ubiquitin E3 ligases in hormonal signaling and development (10).Transfer and covalent ligation of the 76-aa protein ubiquitin to target proteins is a central part of the ubiquitination pathway, involving three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3 is responsible for recruiting specific target proteins for ubiquitination (10). Polyubiquitinated proteins are recognized by the 26S proteasome and degraded. Arabidopsis has two E1 isoforms (11) and at least 37 E2 enzymes (12). In contrast, Ͼ1,300 genes are predicted to encode for E3 ubiquitin ligases (10). The large number and diversity of E3 ligases confer specificity upon the ubiquitination pathway. On the basis of subunit composition and action mode, four E3 types have been described in plants: homology to E6AP C terminus; RING͞U box; a complex of Skp1, CDC53, and F-box protein (SCF); and anaphasepromoting complex. Arabidopsis contains at least 469 predicted RING-domain-containing proteins (13). However, the physiological functions for most of them are not known. The importance of RING proteins in plant responses to cold stress through the ubiquitin͞proteasome pathway has not been investigated. In fact, the involvement of the ubiquitin͞proteasome pathway in cold stress has not been explored in any system.In this study, we have found that cold response in Arabidopsis is attenuated by the proteasome pathway. We demonstrated that the Arabidopsis HOS1 is a functional RING finger protein that has ubiquitin E3 ligase activity. We found that HOS1 physically interacts with ICE1. Both in vitro and in vivo ubiquitination assays showed that HOS1 is required for ICE1 ubiquitination. Furthermore, we discovered that cold induces...

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To study the genetic control of plant responses to cold stress, Arabidopsis thaliana mutants were isolated by a screen for mutations that impair cold-induced transcription of the CBF3-LUC reporter gene. We report here the characterization and cloning of a mutated gene, atnup160-1, which causes reduced CBF3-LUC induction under cold stress. atnup160-1 mutant plants display altered cold-responsive gene expression and are sensitive to chilling stress and defective in acquired freezing tolerance. AtNUP160 was isolated through positional cloning and shown to encode a putative homolog of the animal nucleoporin Nup160. In addition to the impaired expression of CBF genes, microarray analysis revealed that a number of other genes important for plant cold tolerance were also affected in the mutants. The atnup160 mutants flower early and show retarded seedling growth, especially at low temperatures. AtNUP160 protein is localized at the nuclear rim, and poly(A)-mRNA in situ hybridization shows that mRNA export is defective in the atnup160-1 mutant plants. Our study suggests that Arabidopsis AtNUP160 is critical for the nucleocytoplasmic transport of mRNAs and that it plays important roles in plant growth and flowering time regulation and is required for cold stress tolerance.In eukaryotic cells, the genome is enclosed within the nucleus. Nucleocytoplasmic transport of macromolecules across the nuclear membrane occurs through channels formed by nuclear pore complexes (NPCs). Embedded in the double-lipid bilayer nuclear envelope, NPCs form a ringlike structure surrounding a central pore that is believed to facilitate the bidirectional transport of RNAs, proteins, and ribonucleoprotein particles and, at the same time, to allow the diffusion of small molecules and ions across the double membrane (reviewed in reference 4). The overall three-dimensional architecture and transport mechanisms seem to be highly conserved from yeasts to mammals. In Saccharomyces cerevisiae, NPCs are constructed from ϳ30 different nucleoporins with a combined mass of ϳ50 MDa (26). The mammalian NPCs are much larger complexes (ϳ120 MDa) composed of ϳ80 different proteins (10). Although the structural organization of NPCs, the transport mechanism across the channels, and the function of individual nucleoporins have been extensively studied in yeast and vertebrates, very little is known about NPCs in plants.Recently, it was reported that the Arabidopsis thaliana proteins MOS3/SAR3 and SAR1 share high sequence similarities with human nucleoporins Nup96 and Nup160, respectively (25, 34). The putative nucleoporin MOS3/SAR3 was localized at the nuclear rim. The studies suggested that nucleocytoplasmic trafficking plays an important role in plant disease resistance, hormone signaling, and development (25,34). In the present report, we provide evidence that Arabidopsis nucleoporin AtNUP160/SAR1 controls nucleocytoplasmic transport of RNAs and plays important roles in seedling growth, flowering time regulation, and cold stress tolerance.Low temperature is one of...

Ethylene is an important plant growth regulator perceived by membrane-bound ethylene receptors. The ETR1 ethylene receptor is positively regulated by a predicted membrane protein, RTE1, based on genetic studies in Arabidopsis. RTE1 homologs exist in plants, animals and protists, but the molecular function of RTE1 is unknown. Here, we examine RTE1 expression and subcellular protein localization in order to gain a better understanding of RTE1 and its function in relation to ETR1. Arabidopsis plants transformed with the RTE1 promoter fused to the b-glucuronidase (GUS) reporter gene revealed that RTE1 expression partly correlates with previously described sites of ETR1 expression or sites of ethylene response, such as the seedling root, root hairs and apical hook. RTE1 transcript levels are also enhanced by ethylene treatment, and reduced by the inhibition of ethylene signaling. For subcellular localization of RTE1, a functional RTE1 fusion to red fluorescent protein (RFP) was expressed under the control of the native RTE1 promoter. Using fluorescence microscopy, RTE1 was observed primarily at the Golgi apparatus and partially at the endoplasmic reticulum (ER) in stably transformed Arabidopsis protoplasts, roots and root hairs. Next, a functional ETR1 fusion to a 5xMyc epitope tag was expressed under the control of the native ETR1 promoter. Immunohistochemistry of root hairs not only showed ETR1 residing at the ER as previously reported, but revealed substantial localization of ETR1 at the Golgi apparatus. Lastly, we demonstrated the subcellular co-localization of RTE1 and ETR1. These findings support and enhance the genetic model that RTE1 plays a role in regulating ETR1.

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Profilin (PFN) is an ubiquitous, low-M r , actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.

Gaseous phytohormone ethylene affects many aspects of plant growth and development. The ethylene signaling pathway starts when ethylene binds to its receptors. Since the cloning of the first ethylene receptor ETR1 from Arabidopsis, a large number of studies have steadily improved our understanding of the receptors and downstream components in ethylene signal transduction pathway. This article reviews the regulation of ethylene receptors, signal transduction, and the posttranscriptional modulation of downstream components. Functional roles and importance of the ethylene signaling components in plant growth and stress responses are also discussed. Cross-reactions of ethylene with auxin and other phytohormones in plant organ growth will be analyzed. The studies of ethylene signaling in plant growth, development, and stress responses in the past decade greatly advanced our knowledge of how plants respond to endogenous signals and environmental factors.

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development