Philip J. Jensen

The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development.

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.

Investigations into the Role of the Plastidial Peptide Methionine Sulfoxide Reductase in Response to Oxidative Stress in Arabidopsis

Peptidyl Met residues are readily oxidized by reactive oxygen species to form Met sulfoxide. The enzyme peptide Met sulfoxide reductase (PMSR) catalyzes the reduction of Met sulfoxides back to Met. In doing so, PMSR is proposed to act as a last-chance antioxidant, repairing proteins damaged from oxidative stress. To assess the role of this enzyme in plants, we generated multiple transgenic lines with altered expression levels of the plastid form of PMSR (PMSR4). In transgenic plants, PMSR4 expression ranged from 95% to 40% (antisense) and more than 600% (overexpressing lines) of wild-type plants. Under optimal growing conditions, there is no effect of the transgene on the phenotype of the plants. When exposed to different oxidative stress conditions—methyl viologen, ozone, and high light—differences were observed in the rate of photosynthesis, the maximum quantum yield (Fv/Fm ratio), and the Met sulfoxide content of the isolated chloroplast. Plants that overexpressed PMSR4 were more resistant to oxidative damage localized in the chloroplast, and plants that underexpressed PMSR4 were more susceptible. The Met sulfoxide levels in proteins of the soluble fraction of chloroplasts were increased by methyl viologen and ozone, but not by high-light treatment. Under stress conditions, the overexpression of PMSR4 lowered the sulfoxide content and underexpression resulted in an overall increase in content.

Rootstock effects on gene expression patterns in apple tree scions.

Like many fruit trees, apple trees (Malus pumila) do not reproduce true-to-type from seed. Desirable cultivars are clonally propagated by grafting onto rootstocks that can alter the characteristics of the scion. For example, the M.7 EMLA rootstock is semi-dwarfing and reduces the susceptibility of the scion to Erwiniaamylovora, the causal agent of fire blight disease. In contrast, the M.9 T337 rootstock is dwarfing and does not alter fire blight susceptibility of the scion. This study represents a comprehensive comparison of gene expression patterns in scions of the ‘Gala’ apple cultivar grafted to either M.7 EMLA or M.9 T337. Expression was determined by cDNA-AFLP coupled with silver staining of the gels. Scions grafted to the M.9 T337 rootstock showed higher expression of a number of photosynthesis-related, transcription/translation-related, and cell division-related genes, while scions grafted to the M.7 EMLA rootstock showed increased stress-related gene expression. The observed differences in gene expression showed a remarkable correlation with physiological differences between the two graft combinations. The roles that the differentially expressed genes might play in tree stature, stress tolerance, photosynthetic activity, fire blight resistance, and other differences conferred by the two rootstocks are discussed.

Rootstock-regulated gene expression patterns in apple tree scions

Apple trees (Malus x domestica) do not reproduce true-to-type from seed. Therefore, desirable cultivars are clonally propagated by grafting vegetative material onto rootstocks. Although cloned cultivars are genetically identical, rootstocks influence horticulturally important cultivar traits, including tree size, disease resistance, and abiotic stress tolerance. Here, ‘Gala’ scions were grafted to seven different rootstocks that produce a range of tree sizes and grown in a greenhouse. Global gene expression patterns in the scions were compared using a DNA microarray representing 55,230 apple transcripts. Each rootstock triggered a distinct, reproducible scion gene expression pattern. Two thousand nine hundred thirty-four scion transcripts were differentially regulated, by a factor of two or greater, by one or more rootstocks. Transcripts from genes predicted to be involved in responses to stress and biotic and abiotic stimuli were disproportionately represented among the rootstock-regulated transcripts. Microarray data analysis based on tree size identified 116 transcripts whose expression levels were correlated with tree size. The correlation of transcript level with tree size was tested for 14 of these transcripts using quantitative polymerase chain reaction in a population of orchard-grown ‘Mutsu’ cultivar trees grafted onto rootstocks from a breeding population of multiple crosses. Of those tested, transcripts encoding predicted sorbitol dehydrogenase, homeobox-leucine zipper, and hevein-like proteins were confirmed as being expressed at higher levels in larger trees, while a transcript predicted to encode an extensin-like protein was confirmed as being expressed at higher levels in smaller trees. This study illustrates the utility of using rootstock-regulated phenotypes to identify genes potentially associated with horticulturally important traits.

FIBRILLIN4 Is Required for Plastoglobule Development and Stress Resistance in Apple and Arabidopsis

The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO2 fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity.

Oligosaccharides as Immunodeterminants of Erythropoietin for Two Sets of Anti-Carbohydrate Antibodies

Antibodies directed against recombinant erythropoietin have been obtained by immunization of rabbits with the hormone in Freunds complete adjuvant. Two sets of antibodies are present in the serum of the immunized rabbits. The results of oxidation of the erythropoietin with periodate, inhibition of the antibodies with the structural monosaccharide residues of the hormone, and reaction of the antibodies with lectins of known carbohydrate specificity have established the antibodies to be anti-carbohydrate antibodies. These antibodies may be of value as tracking agents for some diseases and should be useful for detecting abuses of the hormone in enhancing performance in athletic competitions.

The C-terminal half of the HrpN virulence protein of the fire blight pathogen Erwinia amylovora is essential for its secretion and for its virulence and avirulence activities.

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.

Identification of apple miRNAs and their potential role in fire blight resistance

MicroRNAs (miRNAs) are key players in multiple biological processes; therefore, analysis and characterization of these small regulatory RNAs is a critical step toward a better understanding of animal and plant biology. In apple (Malus domestica), 200 microRNAs are known, which most probably represent only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of 12 small RNA libraries obtained for fire blight-resistant and fire blight-sensitive trees. In the sequencing results, we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop quantitative PCR (qPCR) and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. The gene ontology (GO) enrichment analysis suggests prominent roles of miRNAs in response to stresses, including pathogen infection. In this study, we identified 116 new and confirmed the expression of 143 already known miRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance. The analyses we performed allowed us to define four apple miRNAs potentially involved in fire blight resistance in apple trees: mdm-miR169a, mdm-miR160e, mdm-miR167b-g, and mdm-miR168a,b. These miRNAs are known to be involved in response to stresses across other plant species, usually by targeting stress response proteins. The relatively low number of candidates may result from the high variance of biological replicates and the fact that stress response miRNAs are usually induced by the stress factors and frequently expressed at a low level, or not expressed at all, in normal conditions. The results of our studies are freely available in an online database at http://lemur.amu.edu.pl/share/apple_miRNAs/.

Mapping in an apple (Malus x domestica) F1 segregating population based on physical clustering of differentially expressed genes.

BackgroundApple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum).ResultsTranscription profiling was performed for 48 individual F1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance.ConclusionsGene expression profiling and trait-associated transcript analysis using an apple F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.