Dale Karlson

Conservation of the cold shock domain protein family in plants

In this paper, we report the widespread occurrence of the nucleic acid-binding cold shock domain (CSD) in plants and identify the first eukaryotic homologs that are nearly identical to bacterial cold shock proteins (CSP). Using Arabidopsis as a model system, we determined that its four unique CSD

Functional dissection of Hydrophilins during in vitro freeze protection

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.

Functional characterization of two cold shock domain proteins from Oryza sativa

Two novel rice cold shock domain (CSD) proteins were cloned and characterized under different stress treatments and during various stages of development. OsCSP1 and OsCSP2 (Oryza sativa CSD protein) encode putative proteins consisting of an N-terminal CSD and glycine-rich regions that are interspersed by 4 and 2 CX(2)CX(4)HX(4)C (CCHC) retroviral-like zinc fingers, respectively. In vivo functional analysis confirmed that OsCSPs can complement a cold-sensitive bacterial strain which lacks four endogenous cold shock proteins. In vitro ssDNA binding assays determined that recombinant OsCSPs are capable of functioning as nucleic acid-binding proteins. Both OsCSP transcripts are transiently up-regulated in response to low-temperature stress and rapidly return to a basal level of gene expression. Protein blot analysis determined that OsCSPs are maintained at a constant level subsequent to a cold treatment lasting over a period of several days. Both the transcript and protein data are in sharp contrast to those previously obtained for winter wheat WCSP1. A time-coursed study through various stages of rice development confirmed that both OsCSP proteins and transcripts are highly accumulated in reproductive tissues and tissues which exhibit meristematic activity.

Arabidopsis cold shock domain proteins: relationships to floral and silique development

Cold shock domain proteins (CSPs) are highly conserved from bacteria to higher plants and animals. Bacterial cold shock proteins function as RNA chaperones by destabilizing RNA secondary structures and promoting translation as an adaptative mechanism to low temperature stress. In animals, cold shock domain proteins exhibit broad functions related to growth and development. In order to understand better the function of CSPs in planta, detailed analyses were performed for Arabidopsis thaliana CSPs (AtCSPs) on the transcript and protein levels using an extensive series of tissue harvested throughout developmental stages within the entire life cycle of Arabidopsis. On both the transcript and protein levels, AtCSPs were enriched in shoot apical meristems and siliques. Although all AtCSPs exhibited similar expression patterns, AtCSP2 was the most abundantly expressed gene. In situ hybridization analyses were also used to confirm that AtCSP2 and AtCSP4 transcripts accumulate in developing embryos and shoot apices. AtCSPs transcripts were also induced during a controlled floral induction study. In vivo ChIP analysis confirmed that an embryo expressed MADS box transcription factor, AGL15, interacts within two AtCSP promoter regions and alters the respective patterns of AtCSP transcription. Comparative analysis of AtCSP gene expression between Landsberg and Columbia ecotypes confirmed a 1000-fold reduction of AtCSP4 gene expression in the Landsberg background. Analysis of the AtCSP4 genomic locus identified multiple polymorphisms in putative regulatory cis-elements between the two ecotypes. Collectively, these data support the hypothesis that AtCSPs are involved in the transition to flowering and silique development in Arabidopsis.

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein

The cold‐induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid‐binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled‐WCSP1 retained its characteristic nucleic acid‐binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA‐binding activity; while a mutant containing the CSD and the first glycine‐rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

Overexpression of AtCSP4 affects late stages of embryo development in Arabidopsis

Eukaryotic cold shock domain proteins are nucleic acid-binding proteins that are involved in transcription, translation via RNA chaperone activity, RNA editing, and DNA repair during tissue developmental processes and stress responses. Cold shock domain proteins have been functionally implicated in important developmental transitions, including embryogenesis, in both animals and plants. Arabidopsis thaliana cold shock domain protein 4 (AtCSP4) contains a well conserved cold shock domain (CSD) and glycine-rich motifs interspersed by two retroviral-like CCHC zinc fingers. AtCSP4 is expressed in all tissues but accumulates in reproductive tissues and those undergoing cell divisions. Overexpression of AtCSP4 reduces silique length and induces embryo lethality. Interestingly, a T-DNA insertion atcsp4 mutant does not exhibit any morphological abnormalities, suggesting that the related AtCSP2 gene is functionally redundant with AtCSP4. During silique development, AtCSP4 overexpression induced early browning and shrunken seed formation beginning with the late heart embryo stage. A 50% segregation ratio of the defective seed phenotype was consistent with the phenotype of endosperm development gene mutants. Transcripts of FUS3 and LEC1 genes, which regulate early embryo formation, were not altered in the AtCSP4 overexpression lines. On the other hand, MEA and FIS2 transcripts, which are involved in endosperm development, were affected by AtCSP4 overexpression. Additionally, AtCSP4 overexpression resulted in up-regulation of several MADS-box genes (AP1, CAL, AG, and SHP2) during early stages of silique development. Collectively, these data suggest that AtCSP4 plays an important role during the late stages of silique development by affecting the expression of several development-related genes.

Phylogenetic Analyses in Cornus Substantiate Ancestry of Xylem Supercooling Freezing Behavior and Reveal Lineage of Desiccation Related Proteins

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.

Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion

The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

AtCSP1 regulates germination timing promoted by low temperature.

An Arabidopsis gene trap line (GT606), which disrupted the AtCSP1 gene, exhibited an early germination phenotype that was affected by stratification treatment. Comparative analysis of GUS expression in seeds at the early germination stage, with or without stratification, demonstrated that AtCSP1 expression was affected by cold temperature. Evaluation of germination assays with varying concentrations of ABA or NaCl revealed a reduced sensitivity of the atcsp1 mutant to both ABA and NaCl. Taken together, these data support the hypothesis that AtCSP1 affects early stages of seed germination subsequent to stratification treatment of seeds.