Lisa J. Rowland

Identification of Dehydrin-Like Proteins Responsive to Chilling in Floral Buds of Blueberry (Vaccinium, section Cyanococcus)

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cyanococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95[deg]C for 15 min. By fractionating bud proteins first by isoelectric point (pl) and then by molecular mass, the pl values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pl value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.

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.

Anaerobic treatment of maize roots affects transcription of Adh1 and transcript stability.

Among the adaptations to stress exhibited by plants is the anaerobic response of roots, induced by submerging roots in water. The response consists of a programmed change in gene expression: proteins produced under aerobic conditions are no longer synthesized but are replaced by approximately 20 so-called anaerobic peptides (M. M. Sachs, M. Freeling, and R. Okimoto, Cell 20:761-767, 1980). The gene for maize alcohol dehydrogenase I (Adh1) is expressed at high levels under such conditions. We report here that changes in alcohol dehydrogenase I RNA levels in anaerobic roots are associated with changes in both transcription rate and transcript stability.

Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions

Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

RAPD-based genetic linkage map of blueberry derived from a cross between diploid species (Vaccinium darrowi and V. elliottii)

An initial genetic linkage map for blueberry has been constructed from over 70 random amplified polymorphic DNA (RAPD) markers that segregated 1∶1 in a testcross population of 38 plants. The mapping population was derived from a cross between two diploid blueberry plants: the F1 interspecific hybrid (Vaccinium darrowi Camp x V. elliottii Chapm.) and another V. darrowi plant. The map currently comprises 12 linkage groups (in agreement with the basic blueberry chromosome number) and covers a total genetic distance of over 950cM, with a range of 3–30cM between adjacent markers. The use of such a map for identifying molecular markers linked to genes controlling chilling requirement and cold hardiness is discussed.

DNA fingerprinting of strawberry (Fragaria × ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers

Forty-one of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the United States and Canada were examined for RAPD (randomly amplified polymorphic DNA) marker polymorphisms using 10mer primers (>50% GC content). A set of 10 primers produced 15 polymorphic fragments ranging in size between 450 and 1200 bp, which were more than sufficient to distinguish among all tested cultivars. Ten of the markers derived from seven primers were absolutely required for distinguishing the cultivars. A DNA fingerprinting table was constructed based on these results. In addition, similarity coefficients were calculated based on RAPD marker data and a dendogram was constructed using the unweighted pair group method of arithmetic averages (UPGMA). These results were compared with known pedigree data for the cultivars. Our results demonstrate that RAPD markers can be used effectively for strawberry cultivar identification.

Anaerobic treatment alters the cell specific expression of Adh-1, Sh, and Sus genes in roots of maize seedlings

SummaryThe expression pattern of Sh and Sus, which encode sucrose synthase isozymes SS1 and SS2, respectively, and Adh-1, which encodes alcohol dehydrogenase 1 (ADH1), was examined in situ in the lower region of the primary root of maize seedlings in response to anaerobiosis. Anaerobic induction of ADH1 RNA was localized to the epidermis and cortex. Induction of Sh was marked by highly elevated SS1 RNA levels in the vascular elements, pith, and epidermis. A significant but less drastic increase in SS protein was found in these same tissues as well as the root cap; the increased level of immunosignal was restricted to cells within approximately 1 cm of the root apex. Sus responded to anaerobic stress with a slight reduction in SS2 RNA and protein levels except in the root cap where SS2 protein, but not SS2 RNA, was induced. Thus, in situ hybridization and/or immunolocalization experiments revealed a unique spatial pattern of expression for each of the three genes in response to anaerobiosis. Furthermore, we conclude that multiple regulatory controls including cell specific post-transcriptional mechanisms modulate SS levels in anaerobically stressed seedlings.

GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency

Abstract Several factors were investigated for their influence on the transfer of an intron-containing β-glucuronidase (GUS) gene into blueberry (Vaccinium spp.) leaf explants during the early stages of Agrobacterium-mediated gene transfer, including days of cocultivation, strain of Agrobacterium tumefaciens, explant age and genotype. The number of GUS-expressing leaf zones and calli were counted immediately and 2 weeks after cocultivation, respectively, to evaluate the gene transfer process. Agrobacterium tumefaciens strain EHA105 (pEHA105/p35SGUS-int) was significantly more effective for transformation than strain LBA4404 (pAL4404/p35SGUSint). Four days of cocultivation with A. tumefaciens strain EHA105 yielded about 50-fold more GUS-expressing zones than 2 days of cocultivation. Significant differences among cultivars were observed for both GUS-expressing leaf zones and calli. For some cultivars, explant age influenced the number of GUS-expressing leaf zones and calli. In most cases, the number of GUS-expressing calli was highest in those cultivars where GUS expression in the leaves was high.

Genetic analysis of freezing tolerance in blueberry (Vaccinium section Cyanococcus)

Abstract An understanding of the genetic control of freezing tolerance (FT) in woody perennials is important for the effective selection and development of plants with a broader climatic adaptation. This study was undertaken to examine the inheritance and gene action of FT in segregating populations of a woody perennial blueberry (Vaccinium, section Cyanococcus). Two backcross populations were derived from interspecific hybrids of the diploid species Vaccinium darrowi andVaccinium caesariense, which are widely divergent in their FT. The bud FTs of uniformly cold acclimated plants of parental, F1, and two backcross populations were evaluated with a laboratory controlled freeze-thaw regime, followed by a visual assessment of injury. FT (LT50) was defined as the temperature causing 50% of the flower buds to be injured. Data indicate that the two parents were homozygous for genes for low or high FT. Freezing-tolerance values of the parental and F1 populations indicate that freeze-sensitivity is a partially dominant trait. Results from reciprocal crosses revealed that there was no significant maternal influence on freezing tolerance. Parental phenotypes were fully recovered in 40–42 plants of each testcross population, suggesting that FT is determined by relatively few genes. The degree of dominance and an analysis of generation means revealed that FT in blueberry is controlled largely by additive gene effects and, to a lesser degree, by dominance gene effects. Testing of various genetic models indicated that FT inheritance can be adequately explained by a simple additive-dominance model; however, two epistatic models involving additive-additive and dominance-dominance interactions also fit the data.

A blackberry (Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers

BackgroundThe recent development of novel repeat-fruiting types of blackberry (Rubus L.) cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST) library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR), and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars.ResultsA cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products.ConclusionThis rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry.