Suppression Subtractive Hybridization Library Research Articles

Insights into the transcriptomics of polyphagy: Bemisia tabaci adaptability to phenylpropanoids involves coordinated expression of defense and metabolic genes

The whitefly Bemisia tabaci is a major generalist agricultural pest of field and horticultural crops world-wide. Despite its importance, the molecular bases of defense mechanisms in B. tabaci against major plant secondary defense compounds, such as the phenylpropanoids, remain unknown. Our experimental system utilized transgenic Nicotiana tabacum plants constitutively expressing the PAP1/AtMYB75 transcription factor which activates relatively specifically the phenylpropanoid/flavonoids biosynthetic pathway. Our study used suppression subtractive hybridization (SSH) and cDNA microarray approaches to compare gene expression between B. tabaci adults subjected to wild-type or transgenic plants for 6 h. A total of 2880 clones from the SSH libraries were sequenced. Both the SSH and cDNA microarray analyses indicated a complex interaction between B. tabaci and secondary defense metabolites produced by the phenylproapnoids/flavonoids pathway, involving enhanced expression of detoxification, immunity, oxidative stress and general stress related genes as well as general metabolism and ribosomal genes. Quantitative real-time PCR revealed significant changes in the expression of several of these genes in response to feeding on artificial diet containing the flavonoids quercetin. The elevated transcriptional activity was not accompanied by reduced reproductive performance, indicating high adaptability of B. tabaci to this large group of plant secondary defense metabolites.

Differential Gene Expression Identified by Suppression Subtractive Hybridization During Late Ripening of Fruit in Oil Palm (Elaeis guineensis Jacq.)

A forward suppression subtractive hybridization (SSH) library was constructed to identify genes involved in fruit ripening in an oil palm (Elaeis guineensis Jacq). The suppression was performed with cDNA extracted from fruits at 12 weeks after anthesis (w.a.a.) as “driver” and at 17 w.a.a. as “tester”. A single-pass sequencing from the 5′-end of 2,112 randomly selected cDNA clones resulted in 2,019 high-quality expressed sequence tags (ESTs) with an average reading length of 383 bp. Clustering of the 2,019 EST sequences using StackPACK showed 20 unigenes consisting of 9 contigs and 11 singletons. Among the edited EST sequences, 1,109 (14 unigenes) had significant matches with protein sequences in the public databases. The matched ESTs were further classified into six putative cellular functions including unclassified proteins (38.0%), cell rescue and defense (33.8%), proteins with binding functions (27.7%), biogenesis of cellular component (0.3%), metabolism (0.1%), and cell cycle (0.1%). The expression levels of the 20 obtained unigenes were analyzed using RT-PCR. At 17 w.a.a., 20 unigenes were significantly up-regulated. The findings in this study will contribute to a better understanding of the molecular mechanisms involved in fruit ripening in oil palm.

Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

Interspecific hybridization in the genus Phaseolus, conducted to introgress desired traits into common bean (Phaseolus vulgaris L.), leads to the abortion of immature embryos, usually at early developmental stages. Little is known about the physiological responses of embryo dysfunction in P. vulgaris during the early stages of embryogenesis and the genes that are involved in these responses. Identification of the genes involved in Phaseolus embryogenesis may provide information that will help to understand the molecular basis of Phaseolus embryo dysfunction. To investigate the genes expressed during Phaseolus seed development, we constructed a suppression subtractive hybridization (SSH) library using cDNA from abortive seed development as the driver and those from normal seed development as tester. The differentially expressed cDNA fragments were identified by differential screening. We identified 72 unique ESTs of which we selected 12 candidates on the basis of their redundancy. These candidates were subjected to a validation procedure based on the study of their expression level by real-time PCR. Sequence analysis revealed that most of the differentially expressed genes are related to metabolism and regulation such as protein synthesis. Some genes also encoded transcription factors. These genes showed high mRNA transcript levels in seed tissues and little or no expression in other tissues (root, stem, flower, leaf, and cotyledon). Seven genes were chosen and their expression profile during seed development in P. vulgaris was analyzed by real-time PCR using RNA preparations originating from different seed development stages. This study revealed hitherto unknown genes putatively involved in dicotyledonous embryogenesis, which serve as a starting point for understanding Phaseolus embryogenesis.

Genetic diversity and comparative analysis of gene expression between Heterorhabditis bacteriophora Az29 and Az36 isolates: Uncovering candidate genes involved in insect pathogenicity

Entomopathogenic nematode Heterorhabditis bacteriophora Az29 and Az36 isolates with different virulence against Popillia unipuncta and soil survival time were isolated from the Azorean archipelago (Portugal) and used for the study. RAPD analysis revealed a very low-level of genetic diversity (GDaxenic Az36 isolateaxenic Az29 isolate=0.2338±0.0541) between axenic Az29 and Az36 isolates, and a relative low-level of diversity (GDAz36 isolateAz29 isolate=0.3366±0.0471) between Az29 and Az36 isolates. To unravel the molecular differences, a suppressive subtractive hybridization library was constructed from the parasitic stage. Assembling 150 high quality ESTs produced 70 singletons and 17 contigs. BLAST analysis revealed that 48 ESTs showed significant similarity to known protein and 39 ESTs had no significant hits in the database, perhaps representing novel genes. Functional annotation revealed some of these genes to be involved in metabolism, cellular process and signaling, information storage and processing, stress response and host–parasite interactions. Genes with a role in the parasitism process were identified including lectin, metalloprotease, enolase, chitinase, surface-associated antigen, and as well as genes (aquaporin, Hsp70A, Hsp10 and Hsp20) essential for stresses tolerance. The work described here provides the molecular data necessary for investigating the fundamental molecular aspects of host–parasite interactions. Future investigations should be focused on determining the molecular mechanism of those genes in entomopathogenic nematode life cycle.

Identification of differentially expressed genes in response to infection of a mild Citrus tristeza virus isolate in Citrus aurantifolia by suppression subtractive hybridization

Citrus tristeza virus (CTV) is the most serious viral pathogen of citrus. Pathogenicity differentiation among CTV isolates has been found in some countries. In order to better understand the responses of Mexican lime ( Citrus aurantifolia) to the infection of a mild CTV isolate (CTV-N4), we constructed two suppression subtractive hybridization (SSH) libraries. A total of 589 expressed sequence tags (ESTs) were screened from two SSH libraries by dot blot analysis. Sequencing results showed that these ESTs represented 202 genes, of which 111 and 91 genes were up-regulated and down-regulated, respectively. The differential expression of 10 out of 13 selected genes was confirmed by quantitative real-time RT-PCR (qRT-PCR). All differently expressed genes were functionally categorized into eight groups, of which the majority was associated with stress and disease defenses. The transcriptional levels of three genes in lime plants with the infection of a mild isolate CTV-N4 and a severe isolate CTV-N21 were comparatively analyzed by qRT-PCR over a 120 days time course. Results indicated that transcriptional changes of three studied genes were more greatly induced by CTV-N21 than those induced by CTV-N4. The obtained results provided novel information on genes potentially involved in citrus responses to CTV infection.

An Improved Protocol for the Isolation of RNA from Roots of Tea (Camellia sinensis (L.) O. Kuntze)

Tea, a beverage crop, is a rich source of polyphenols and polysaccharides which greatly attribute to its importance. However, oxidation and precipitation of these compounds during nucleic acids extraction is a limitation to molecular biology and genomic studies. On isolation of total RNA from root tissue using established protocols, difficulties were encountered in terms of purity and quantity of isolated RNA or some of the methods were time-consuming and also yields were low. The present communication combines a phenol-based RNA isolation protocol with a cetyltrimethylammonium bromide-based procedure with appropriate modifications. This protocol successfully isolated RNA from tap root tissue in 2-3 h as compared with 16 h reported by the previous method. Also, RNA yield was higher by more than fourfold. The RNA isolated by this protocol was successfully used for downstream applications such as RT-PCR and the construction of suppression subtractive hybridization library. The developed protocol worked well with other plant tissue with high polyphenols and polysaccharides contents.

Screening and identification of specific markers for bladder transitional cell carcinoma from urine urothelial cells with suppressive subtractive hybridization and cDNA microarray

The objective of this study was to screen and identify differentially expressed genes in invasive bladder transitional cell carcinoma (BTCC). Voided urine samples were collected from consecutive patients with BTCC and patients under surveillance for bladder cancer recurrence; voided urine samples from patients with non-malignant diseases served as control. We identified the differentially expressed genes by comparing urine samples of bladder carcinoma to that of the control group with suppressive subtractive hybridization (SSH) and cDNA microarray. The differentially expressed genes were verified by quantitative real-time polymerase chain reaction (QPCR). From the 762 white colonies, a total of 449 positive clones were obtained in which 112 were found to be upregulated in BTCC. Sequencing and homology analysis were performed for these 112 clonies. The detection rates of some known genes (including IGF-1, human telomerase reverse transcriptase [hTERT], bladder cancer specific nuclear matrix protein 4 [BLCA-4] and homeobox A13 [HOXA13]) for BTCC at the Ta, T1 and >T1 stages were 48%, 90% and 100%, respectively, with a specificity of 85%. The test specificity was 80% for the 30 control patients with urinary tract infections. The combination of BLCA-4 and HOXA13 could distinguish between low- and high-grade tumours, with specificity and sensitivity of 80%. We successfully constructed a reliable SSH library of BTCC and found that combination detection insulin-like growth factor 1 (IGF-1), hTERT, BLCA-4 and HOXA13 genes could help to evaluate BTCC at different stages.

Expressions of KLF1, FOXO3a, HOOK3 and MIER1 genes Are Associated with Fetal Hemoglobin Levels in Hereditary Persistence of Fetal Hemoglobin,

Abstract 3195The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies.Support by FAPESP, CNPq.and INCTS Disclosures:No relevant conflicts of interest to declare.

Light-induced expression of genes involved in phenylpropanoid biosynthetic pathways in callus of tea ( Camellia sinensis (L.) O. Kuntze)

Tea ( Camellia sinensis (L.) O. Kuntze) is a commercially important crop that is valued for its secondary metabolites. Light is an important environmental parameter that regulates plant growth and development and influences the phenylpropanoid metabolism in plants. To investigate the molecular mechanism by which light regulates phenylpropanoid metabolism, we established light-induced suppression subtractive hybridization (SSH) cDNA libraries of tea calli. A total of 265 clones from the library were selected, sequenced, and analyzed in this study. Nine diverse ESTs involved in phenylpropanoid biosynthesis were detected in the library. A new CsDFR gene ( CsDFR2), higher increment of the expression activated by light than the previously reported CsDFR gene ( CsDFR1), was cloned. The key phenylpropanoid compounds and representative genes expression analysis implied that light could be effective for activation of the biosynthesis of phenylpropanoids. Compared to the darkness control, levels of lignins, catechins, and PAs were increased 3.46, 3.00, and 1.21-fold, in light-induced calli, respectively. And lignin biosynthesis genes, involved in CCoAOMT, HCT and CCR, were identified in the light-induced SSH library. Therefore it was assumed that lignins might be the main phenylpropanoid metabolites activated by light in tea calli. In addition, our researches found that catechins, as the main secondary metabolites, significantly decreased in the tea calli compared to those in tea mature leaves, While PAs (polymer of catechins) in calli did not decrease compared to mature leaves. The data suggest that polymerization reaction might be the main pathway of flavonoid metabolism in tea callus. The SSH library established in this study represents a valuable resource for better understanding the mechanisms of light-induced secondary metabolism in tea plants.

Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures

Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.

Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization

Trifoliate orange ( Poncirus trifoliata (L.) Raf.) is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular events underlying the acclimation is still limited so far. In this study, forward (4 °C over 25 °C) and reverse (25 °C over 4 °C) suppression subtractive hybridization (SSH) libraries were constructed in order to identify the genes involved in cold acclimation in trifoliate orange. After reverse northern blotting analysis and sequencing, a total of 105 and 117 non-redundant differentially expressed sequence tags (ESTs) were obtained from the forward and reverse libraries, respectively. Blast2go analysis revealed that 91 ESTs, 31 from the forward library and 60 from the reverse library, displayed significant sequence homology to the genes with known or putative functions. They were categorized into various functional groups, including catalytic activity, binding protein, structural molecule, enzyme regulator, molecular transducer, electron carrier, and transport activity/transcription regulation. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction was consistent with the results of differential screening. In addition, time-course expression patterns of the genes further confirmed that they were responsive to low temperature treatment. Among the genes of known functions, many are related to maintenance of cell wall integrity, adjustment of osmotic potential and maintenance of reactive oxygen species homeostasis, implying that these physiological processes might be of paramount significance in rendering protective mechanisms against the low temperature stress. The data presented here gain an insight into the molecular changes underlying the cold acclimation of trifoliate orange, and the results can be of reference for unraveling candidate genes that hold great potential for genetic engineering in an effort to create novel germplasms with enhanced cold stress tolerance.

Characterization of upregulated genes associated with high phosphorus accumulation in cucumber

Excessive application of phosphorus (P)-rich manures to agricultural lands often results in P-accumulation in soils leading to water pollution through runoffs and leaching. Use of suitable plant species that can extract and sequester excess P from soil into their biomass is an effective method of remediation of P-contaminated soils. Knowledge on the molecular responses of plants to high P-accumulation and tolerance is lacking. Therefore, a suppression subtractive hybridization (SSH) strategy was employed to identify and elucidate the pattern of gene expression related to P-tolerance and accumulation in cucumber (Cucumis sativus L.), a P-accumulator plant. RNA isolated from cucumber grown in high P was used for 'tester' cDNA synthesis and SSH library preparation. A total of 63 cDNAs were identified as showing upregulated expression in this plant in response to high P. No putative function could be assigned to 7 (11%) of the 63 upregulated high P-modulated genes and 11 expressed sequence tags (ESTs) (17%) did not match database entries. The remaining 45 ESTs were grouped into five functional classes. The majority of these ESTs belonged to three groups: 'metabolism', 'protein synthesis/degradation and signaling' and 'cell structure/cell wall'. Only six 'stress/defense'-related ESTs were identified from this library. The results of reverse northern blot analysis was further confirmed and validated through semi-quantitative RT-PCR carried out with representative ESTs identified in this study. The research reported here may contribute to a preliminary understanding of the high P-related gene expression in this P-accumulating plant.

Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by <I>Puccinia striiformis</I> Westend. f. sp. <I>tritici</I>

To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

Identification and characterization of differentially expressed genes from tobacco roots after decapitation

Nicotine is the major alkaloid in tobacco and its biosynthesis is regulated by a variety of factors. Topping, as an important agronomic factor, can induce the nicotine biosynthesis increase. Some key nicotine biosynthesis-related genes and the framework of nicotine biosynthesis pathway have been well studied, but the details of nicotine biosynthesis pathway are not well understood now. To investigate the genes expressed after tobacco topping, we constructed a suppression subtractive hybridization library using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester. The insert size of positive clones was 200–1,000 bp confirmed by PCR. After differential screening, 560 significantly differently expressed clones among 1,950 positive clones were acquired, sequenced and 273 high quality expressed sequence tags (ESTs) were acquired. The results of nucleotide blast homological analysis indicated that these ESTs mainly involved in alkaloid biosynthesis (4%), plant hormone metabolism (3%), signaling/transcription (18%), stress/defense (32%), protein metabolism (9%), carbon metabolism (6%), other metabolism (15%) and function unknown (13%). The expression of selected genes was analyzed by reverse transcription polymerase chain reaction and RNA gel blot hybridization, and the result indicated that their transcription amount increased after tobacco topping. NtNAC-R1was in silico cloned, and the expression level of NtNAC-R1 increased at 12 and 24 h in tobacco roots after topping, which indicated that NtNAC-R1 may play an important role in the signal transduction after tobacco topping. In addition to many previously reported nicotine biosynthesis-related genes, some new genes, such as transcription factors related to nicotine biosynthesis/regulation and the members of plant hormone pathway, were discovered in our library. The results contribute new data to the list of possible candidate genes involved in nicotine biosynthesis and regulation.

English

In this study, forward and reverse suppression subtractive hybridization (SSH) libraries were constructed between female and male gametophyte of Gracilaria lemaneiformis to isolate genes differentially expressed between gametophytes. Dot-blots were performed to screen 384 colonies randomly selected from each subtracted libraries. Partial gene (designated as GMF-01) which is female gametophyte-specific was selected to isolate its cDNA full length sequences and to be characterized. Then we got its cDNA full length sequences using SMART-RACE technic. Sequence analysis showed that the open reading frame (ORF) of GMF-01 is 1002 bp long with a GC content of 47.7%, encoding 333 amino acids. GMF-01 does not have significant match in the databases when examined on NCBI website. Amino acid sequence analysis showed that the theoretical pI of this translated protein is 7.92, while the instability index is computed to be 43.61, which has classified the protein as unstable. Results of prediction showed 45.05% of its secondary structure has random coil and sub-cellular location prediction indicated that it is probably a extracellular protein.

Gene expression associated with vegetative incompatibility in Amylostereum areolatum

In filamentous fungi, vegetative compatibility among individuals of the same species is determined by the genes encoded at the heterokaryon incompatibility (het) loci. The hyphae of genetically similar individuals that share the same allelic specificities at their het loci are able to fuse and intermingle, while different allelic specificities at the het loci result in cell death of the interacting hyphae. In this study, suppression subtractive hybridization (SSH) followed by pyrosequencing and quantitative reverse transcription PCR were used to identify genes that are selectively expressed when vegetatively incompatible individuals of Amylostereum areolatum interact. The SSH library contained genes associated with various cellular processes, including cell-cell adhesion, stress and defence responses, as well as cell death. Some of the transcripts encoded proteins that were previously implicated in the stress and defence responses associated with vegetative incompatibility. Other transcripts encoded proteins known to be associated with programmed cell death, but have not previously been linked with vegetative incompatibility. Results of this study have considerably increased our knowledge of the processes underlying vegetative incompatibility in Basidiomycetes in general and A. areolatum in particular.

Mytilus galloprovincialis Myticin C: A Chemotactic Molecule with Antiviral Activity and Immunoregulatory Properties

Previous research has shown that an antimicrobial peptide (AMP) of the myticin class C (Myt C) is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH) libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme). Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped). Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.

Isolation and characterization of genes expressed differently in mature fruits of ‘redfield’ and ‘greensleeves’ apples

Anthocyanins are responsible for most of the bright red and blue colors found in higher plants. Anthocyanin accumulation is regulated by MYB transcription factors. Some varieties of apple indicate anthocyanin accumulation with a red color in the skin and, at times, even in the cortex core, but other varieties without anthocyanin production have green-colored skin indicative of chlorophyll accumulation. To obtain gene expression profiles from the apple cultivars with different flesh and skin colors, 2 apple cultivars, the red-colored ‘Redfield’ and the green-colored ‘Greensleeves’, were used. A cDNA suppression subtractive hybridization library was established and analyzed. Six genes encoding methallothionein-like protein, chalcone isomerase, dirigent-like protein, brassinosteroid-6-oxidase, an unnamed protein product, and some unknown proteins were selected, and their expression was confirmed in the ‘Redfield’ cultivar through virtual northern blot analysis. In the ‘Greensleeves’ cultivar, 5 genes encoding gibberellins-regulated protein, a hypothetical 23.5 KD protein, anthranilate N-hydroxycinnamoyl/benzoyltransferase, and isoflavone reductase were chosen, and their upregulated transcriptional levels were identified. These selected genes were differently expressed in each apple cultivar, suggesting that these genes directly or indirectly regulate anthocyanin accumulation.

EST profiling of resistant and susceptible Hevea infected by Microcyclus ulei

In this report, we assess by suppression subtractive hybridization (SSH) the biological process of the South American leaf blight (SALB) that is an important disease of rubber tree caused by Microcyclus ulei (P. Hem.) v. Arx. Leaves from MDF180 (a partially resistant cultivar from Peruvian origin) and PB314 (a susceptible cultivar) were inoculated with M. ulei. Leaf fragments were collected from 6h post-infection (hpi) to 58 days post-infection (dpi) and five cDNA libraries enriched for genes induced by M. ulei in order to identify up-regulated genes at the different stages of the host-parasite interaction. We sequenced 8027 expressed sequence tags (EST). After control of their quality, trimming and assembling, we obtained 1165 singlets and 458 contigs. The EST redundancy within each SSH library varied from 23% to 87%. Based on sequence similarity 49% of the sequences could be assigned to functional categories, whereas 21% corresponded to unknown function and 30% did not show any significant similarity with other sequences. The profile of gene expression was different for each library and gave a comprehensive picture of the transcriptome dynamics of rubber tree in response to M. ulei throughout the infection process. Most of the ESTs described in this study (80%) are specific to leaf tissues since they did not show any homology with ESTs previously identified in latex transcriptome. The occurrence of ESTs whose functional annotation was related to “stress” or “defence” was compared between libraries and their specific function is discussed in relation to the corresponding stage of leaf lesions and to histological observations. As far as we known, this is the first report on ESTs from Hevea brasiliensis collected during a biotic stress. It provides a significant contribution to the understanding of the molecular mechanism of the resistance and the susceptibility to South American leaf blight and it represents a new molecular resource for the development of polymorphic markers and marker-assisted selection.

Identification and validation of differentially expressed transcripts in a hepatocyte model of cold-induced glycerol production in rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) avoid freezing by producing antifreeze protein (AFP) and accumulating glycerol. Glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature. Hepatocytes were isolated from the livers of fish acclimated to 8°C. Cells were incubated at warm (8°C; nonglycerol accumulating) or cold (0.4°C; glycerol accumulating) temperature over a 72-h time course. Reciprocal suppression subtractive hybridization libraries enriched for cold-responsive transcripts were constructed at 72 h. Microarray analyses using a 16K salmonid cDNA array were performed at 24, 48, and 72 h. Expression of type II AFP and 21 carbohydrate, amino acid, or lipid metabolism-related transcripts were validated using quantitative RT-PCR. Type II AFP transcript levels were not directly temperature related. In cold cells, levels of the glucose synthesis transcript were transiently higher. Increased glycerol production was not associated with increased phosphofructokinase or cytosolic glycerol-3-phosphate dehydrogenase transcript levels. Levels of transcripts (phosphoenolpyruvate carboxykinase, mitochondrial malate dehydrogenase, alanine aminotransferase, glutamate dehydrogenase, and aquaglyceroporin 9) associated with mobilization of amino acids to fuel glycerol accumulation were all transiently higher, suggesting a common regulatory mechanism. In cold compared with warm cells, pyruvate dehydrogenase kinase [an inhibitor of pyruvate dehydrogenase (PDH)] transcript levels were 20-fold higher. Potent inhibition of PDH would direct pyruvate and oxaloacetate derived from amino acids to glycerol, as opposed to oxidation via the citric acid cycle. Levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were higher following cold incubation. Finally, this study also presents the novel finding of increased glutamine synthetase transcript levels in response to low temperature.