Stephen L. Krebs

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°C) leaves, and the other from summer-collected (non-acclimated, NA; leaf freezing tolerance −7°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.

Affinity purification and characterization of a β-1,3-glucanase from celery

Abstract β-1,3-glucanase was purified in a single column step from crude root extracts of celery, Apium graveolens L. using a novel affinity chromatography procedure. The β-1,3-glucanase was bound to an insoluble anhydroglucose substrate (pachyman) and then eluted by the addition of a soluble substrate (laminarin). The celery enzyme has an isoelectric point of 4.1, an estimated molecular mass of 35 kDa, maximum endo-β-1,3-glucanase activity at pH 5.2 and a turnover rate of t 2800 nmol substrate/nmol enzyme/min with laminarin as substrate. The purified enzyme partially hydrolyzed isolated cell walls of the celery with pathogen, Fusarium oxysporum Schlect f. sp. apii (R. Nels. and Sherb.) Snyd. and Hans.; its activity was substantially increased by the addition of crude root extract containing chitinase. Chitinase activity on the fungal cell walls was not enhanced by supplementing crude root extract with additional purified β-1,3-glucanase.

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 Tmax, 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 Tmax 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 Tmax 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)—Tmax of 4-year-old ramets was significantly lower than the Tmax 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.