The relationship of cold acclimation and extracellular ice formation to winter thermonasty in two Rhododendron species and their F1 hybrid.

PREMISE: Thermonastic leaf movements in evergreen Rhododendron species have been used to study plant strategies for winter photoprotection. To add to the current fundamental understanding of this behavior, we addressed the following questions: (1) Is the cold-acclimated (CA) state necessary for thermonasty, and do cold-induced leaf movements also occur in non-acclimated (NA) plants? (2) Which of the two movements, leaf rolling versus curling, is more responsive to freezing, if any, in a non-thermonastic species? (3) What is the temporal relationship between extracellular freezing and thermonasty? (4) What genetic inferences can be drawn from leaf movement in an F1 hybrid relative to its parents? METHODS: A temperature-controlled, gradual cooling regime was used to quantify freeze-induced leaf movements. Infrared thermography was used to confirm extracellular ice-formation in leaves. RESULTS: Both NA and CA plants of thermonastic species exhibited thermonasty, but leaf rolling/curling increased significantly in CA plants. In the cold-acclimated condition, a non-thermonastic species showed almost no rolling during freezing, while the thermonastic species and F1 hybrid did, the latter exhibiting a response intermediate to the parents. Freezing-induced leaf curling in the non-thermonastic species and the F1 hybrid was equivalent and significantly less than the degree of curling in the thermonastic species. CONCLUSIONS: Milder thermonasty in NA than CA leaves could be associated with differential anisotropy in the rolling forces and/or response of aquaporins to freezing. Leaf movements in the hybrid suggest that leaf rolling and curling are additive and dominant genetic traits, respectively. Infrared thermography confirms that ice formation in tissues precedes cold-induced thermonasty in R. catawbiense.

Phylogenetic analysis and seasonal cold acclimation-associated expression of early light-induced protein genes of Rhododendron catawbiense.

The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

RcDhn5, a cold acclimation-responsive dehydrin from Rhododendron catawbiense rescues enzyme activity from dehydration effects in vitro and enhances freezing tolerance in RcDhn5-overexpressing Arabidopsis plants.

Dehydrins (DHNs) are typically induced in response to abiotic stresses that impose cellular dehydration. As extracellular freezing results in cellular dehydration, accumulation of DHNs and development of desiccation tolerance are believed to be key components of the cold acclimation (CA) process. The present study shows that RcDhn5, one of the DHNs from Rhododendron catawbiense leaf tissues, encodes an acidic, SK(2) type DHN and is upregulated during seasonal CA and downregulated during spring deacclimation (DA). Data from in vitro partial water loss assays indicate that purified RcDhn5 protects enzyme activity against a dehydration treatment and that this protection is comparable with acidic SK(n) DHNs from other species. To investigate the contribution of RcDhn5 to freezing tolerance (FT), Arabidopsis plants overexpressing RcDhn5 under the control of 35S promoter were generated. Transgenic plants exhibited improved 'constitutive' FT compared with the control plants. Furthermore, a small but significant improvement in FT of RcDhn5-overexpressing plants was observed after 12 h of CA; however, this gained acclimation capacity was not sustained after a 6-day CA. Transcript profiles of cold-regulated native Arabidopsis DHNs (COR47, ERD10 and ERD14) during a CA time-course suggests that the apparent lack of improvement in cold-acclimated FT of RcDhn5-overexpressing plants over that of wild-type controls after a 6-day CA might have been because of the dilution of the effect of RcDhn5 overproduction by a strong CA-induced expression of native Arabidopsis DHNs. This study provides evidence that RcDhn5 contributes to freezing stress tolerance and that this could be, in part, because of its dehydration stress-protective ability.

Dehydrin variability among rhododendron species: a 25-kDa dehydrin is conserved and associated with cold acclimation across diverse species.

• Here we examine the accumulation pattern of dehydrins in non- vs cold-acclimated leaves of 21 species comprising two divergent groups of Rhododendron, Subgenus Hymenanthes and Subgenus Rhododendron. Individuals from five other Ericaceous genera were also evaluated in the same way. Quantitative comparisons of cold-inducibility of a 25-kDa dehydrin and cold acclimation ability in six Rhododendron species were also performed. • Leaf freezing tolerance assay and dehydrin detection and quantification were performed as previously described. • Eleven dehydrins, ranging from 25- to 73-kDa, were observed among the 21 species, and most were more abundant in winter-collected leaves than in summer-collected leaves. One dehydrin, a 25-kDa protein, was uniquely conserved across most (95%) of the species surveyed, and was absent only in R. brookeanum, a tropical species that may not be capable of cold acclimation. The 25-kDa dehydrin was also identified in Kalmia, a genus closely related to Rhododendron but not in four other less related Ericaceous genera. Comparison of dehydrin profiles in non- and cold-acclimated leaf tissue from six species (three very hardy, and three less hardy, species) indicated a close association (R2  = 0.95) between relative changes in leaf freezing tolerance and 25-kDa dehydrin accumulation. • The taxonomic and physiological comparisons suggest a central, but as yet unknown, function for the 25-kDa dehydrin in protecting rhododendron leaves from freezing injury.

Cold hardiness increases with age in juvenile Rhododendron populations.

Winter survival in woody plants is controlled by environmental and genetic factors that affect the plant's ability to cold acclimate. Because woody perennials are long-lived and often have a prolonged juvenile (pre-flowering) phase, it is conceivable that both chronological and physiological age factors influence adaptive traits such as stress tolerance. This study investigated annual cold hardiness (CH) changes in several hybrid Rhododendron populations based on T max, an estimate of the maximum rate of freezing injury (ion leakage) in cold-acclimated leaves from juvenile progeny. Data from F2 and backcross populations derived from R. catawbiense and R. fortunei parents indicated significant annual increases in T max ranging from 3.7 to 6.4°C as the seedlings aged from 3 to 5 years old. A similar yearly increase (6.7°C) was observed in comparisons of 1- and 2-year-old F1 progenies from a R. catawbiense × R. dichroanthum cross. In contrast, CH of the mature parent plants (>10 years old) did not change significantly over the same evaluation period. In leaf samples from a natural population of R. maximum, CH evaluations over 2 years resulted in an average T max value for juvenile 2- to 3-year-old plants that was 9.2°C lower than the average for mature (~30 years old) plants. A reduction in CH was also observed in three hybrid rhododendron cultivars clonally propagated by rooted cuttings (ramets)-T max of 4-year-old ramets was significantly lower than the T max estimates for the 30- to 40-year-old source plants (ortets). In both the wild R. maximum population and the hybrid cultivar group, higher accumulation of a cold-acclimation responsive 25 kDa leaf dehydrin was associated with older plants and higher CH. The feasibility of identifying hardy phenotypes at juvenile period and research implications of age-dependent changes in CH are discussed.

Comparative analysis of expressed sequence tags from cold-acclimated and non-acclimated leaves of Rhododendron catawbiense Michx.

An expressed sequence tag (EST) analysis approach was undertaken to identify major genes involved in cold acclimation of Rhododendron, a broad-leaf, woody evergreen species. Two cDNA libraries were constructed, one from winter-collected (cold-acclimated, CA; leaf freezing tolerance -53 degrees C) leaves, and the other from summer-collected (non-acclimated, NA; leaf freezing tolerance -7 degrees C) leaves of field-grown Rhododendron catawbiense plants. A total of 862 5'-end high-quality ESTs were generated by sequencing cDNA clones from the two libraries (423 from CA and 439 from NA library). Only about 6.3% of assembled unique transcripts were shared between the libraries, suggesting remarkable differences in gene expression between CA and NA leaves. Analysis of the relative frequency at which specific cDNAs were picked from each library indicated that four genes or gene families were highly abundant in the CA library including early light-induced proteins (ELIP), dehydrins/late embryogenesis abundant proteins (LEA), cytochrome P450, and beta-amylase. Similarly, seven genes or gene families were highly abundant in the NA library and included chlorophyll a/b-binding protein, NADH dehydrogenase subunit I, plastidic aldolase, and serine:glyoxylate aminotransferase, among others. Northern blot analyses for seven selected abundant genes confirmed their preferential expression in either CA or NA leaf tissues. Our results suggest that osmotic regulation, desiccation tolerance, photoinhibition tolerance, and photosynthesis adjustment are some of the key components of cold adaptation in Rhododendron.