In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

In silico prediction of gene expression patterns in Citrus flavedo

Out of the 18,942 flavedo expressed sequences (clusters plus singletons) in Citrus sinensis from the Citrus EST Project (CitEST), 25 were statistically supported to be differentially expressed in this tissue after a double in silico hybridization strategy against leaf-, flower-, and bark-derived ESTs. Five of them, two terpene synthases and three O-methyltransferases, are absent in the other citrus tissues with concomitant 2x2 statistics, supporting the hypothesis that they are putative flavedo-specific expressed sequences. The pattern of these differentially expressed sequences during fruit development suggests that most of them are developmentally regulated. Some expressed gene products, including a putative germin-like protein highly expressed in flavedo, are shown to be promising candidates for further characterization. In addition to promoter seeking, this kind of analysis can lead to gene discovery, tissue-specific and tissue-enriched expression pattern predictions (as shown herein) and can also be adopted as an in silico first, and probably reliable approach, for detecting expression profiles from EST sequencing efforts before experimental validation is available or for heuristically guiding that validation.

In silico analysis of ESTs from roots of Rangpur lime (Citrus limonia Osbeck) under water stress

CitEST project resulted in the construction of cDNA libraries from different Citrus sp. tissues under various physiological conditions. Among them, plantlets of Rangpur lime were exposed to hydroponic conditions with and without water stress using PEG6000. RNA from roots was obtained and generated a total of 4,130 valid cDNA reads, with 2,020 from the non-stressed condition and 2,110 from the stressed set. Bioinformatic analyses measured the frequency of each read in the libraries and yielded an in silico transcriptional profile for each condition. A total of 40 contigs were differentially expressed and allowed to detect up-regulated homologue sequences to well known genes involved in stress response, such as aquaporins, dehydrin, sucrose synthase, and proline-related synthase. Some sequences could not be classified by using FunCat and remained with an unknown function. A large number of sequences presented high similarities to annotated genes involved with cell energy, protein synthesis and cellular transport, suggesting that Rangpur lime may sustain active cell growth under stressed condition. The presence of membrane transporters and cell signaling components could be an indication of a coordinated morphological adaptation and biochemical response during drought, helping to explain the higher tolerance of this rootstock to water stress.

PR gene families of citrus: their organ specific-biotic and abiotic inducible expression profiles based on ESTs approach

In silico expression profiles, of the discovered 3,103 citrus ESTs putatively encoding for PR protein families (PR-1 to PR-17), were evaluated using the Brazil citrus genome EST CitEST/database. Hierarchical clustering was displayed to identify similarities in expression patterns among citrus PR-like gene families (PRlgf) in 33 selected cDNA libraries. In this way, PRlgf preferentially expressed by organ and citrus species, and library conditions were highlighted. Changes in expression profiles of clusters for each of the 17 PRlgf expressed in organs infected by pathogens or drought-stressed citrus species were displayed for relative suppression or induction gene expression in relation to the counterpart control. Overall, few PRlgf showed expression 2-fold higher in pathogen-infected than in uninfected organs, even though the differential expression profiles displayed have been quite diverse among studied species and organs. Furthermore, an insight into some contigs from four PRlgf pointed out putative members of multigene families. They appear to be evolutionarily conserved within citrus species and/or organ- or stress-specifically expressed. Our results represent a starting point regarding the extent of expression pattern differences underlying PRlgf expression and reveal genes that may prove to be useful in studies regarding biotechnological approaches or citrus resistance markers.

Analysis of expressed sequence tags from Citrus sinensis L. Osbeck infected with Xylella fastidiosa

In order to understand the genetic responses resulting from physiological changes that occur in plants displaying citrus variegated chlorosis (CVC) symptoms, we adopted a strategy of comparing two EST libraries from sweet orange [Citrus sinensis (L.) Osbeck]. One of them was prepared with plants showing typical CVC symptoms caused by Xylella fastidiosa and the other with non-inoculated plants. We obtained 15,944 ESTs by sequencing the two cDNA libraries. Using an in silico hybridization strategy, 37 genes were found to have significant variation at the transcriptional level. Within this subset, 21 were up-regulated and 16 were down-regulated in plants with CVC. The main functional categories of the down-regulated transcripts in plants with CVC were associated with metabolism, protein modification, energy and transport facilitation. The majority of the up-regulated transcripts were associated with metabolism and defense response. Some transcripts associated with adaptation to stress conditions were up-regulated in plants with CVC and could explain why plants remain alive even under severe water and nutritional stress. Others of the up-regulated transcripts are related to defense response suggesting that sweet orange plants activate their defense machinery. The genes associated with stress response might be expressed as part of a secondary response related to physiological alterations caused by the infection.

Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

The Citrus ESTs Sequencing Project (CitEST) conducted at Centro APTA Citros Sylvio Moreira/IAC has identified and catalogued ESTs representing a set of citrus genes expressed under relevant stress responses, including diseases such as citrus variegated chlorosis (CVC), caused by Xylella fastidiosa. All sweet orange (Citrus sinensis L. Osb.) varieties are susceptible to X. fastidiosa. On the other hand, mandarins (C. reticulata Blanco) are considered tolerant or resistant to the disease, although the bacterium can be sporadically detected within the trees, but no disease symptoms or economic losses are observed. To study their genetic responses to the presence of X. fastidiosa, we have compared EST libraries of leaf tissue of sweet orange Pêra IAC (highly susceptible cultivar to X. fastidiosa) and mandarin ‘Ponkan’ (tolerant) artificially infected with the bacterium. Using an in silico differential display, 172 genes were found to be significantly differentially expressed in such conditions. Sweet orange presented an increase in expression of photosynthesis related genes that could reveal a strategy to counterbalance a possible lower photosynthetic activity resulting from early effects of the bacterial colonization in affected plants. On the other hand, mandarin showed an active multi-component defense response against the bacterium similar to the non-host resistance pattern.

Differential expression of genes identified from Poncirus trifoliata tissue inoculated with CTV through EST analysis and in silico hybridization

Citrus is the most important fruit crop in Brazil and Citrus tristeza virus (CTV) is considered one of the most important pathogens of citrus. Most citrus species and varieties are susceptible to CTV infection. However, Poncirus trifoliata, a close relative of citrus, is resistant to the virus. In order to better understand the responses of citrus plants to the infection of CTV, we constructed expressed sequence tag (EST) libraries with tissues collected from Poncirus trifoliata plants, inoculated or not with Citrus tristeza virus at 90 days after inoculation, grafted on Rangpur lime rootstocks. We generated 17,867 sequence tags from Poncirus trifoliata inoculated (8,926 reads) and not (8,941 reads) with a severe CTV isolate. A total of 2,782 TCs (Tentative Consensi sequences) were obtained using both cDNA libraries in a single clusterization procedure. By the in silico hybridization approach, 289 TCs were identified as differentially expressed in the two libraries. A total of 121 TCs were found to be overexpressed in plants infected with CTV and were grouped in 12 primary functional categories. The majority of them were associated with metabolism and defense response. Some others were related to lignin, ethylene biosynthesis and PR proteins. In general, the differentially expressed transcripts seem to be somehow involved in secondary plant response to CTV infection.

Differentially expressed stress-related genes in the compatible citrus-Citrus leprosis virus interaction

Leprosis, caused by Citrus leprosis virus, cytoplasmic type (CiLV-C), is the main viral disease in the Brazilian citrus industry. This occurs because of the widespread source of inoculum and the year-round presence of the vector, the tenuipalpid mite Brevipalpus phoenicis, in citrus plants. In addition, while some Citrus species are resistant to CiLV-C, C. sinensis, the main cultivated species in the country, is extremely susceptible to the disease. The main objective of this work was to identify genes in C. sinensis cv. Pêra plants that were differentially expressed after the host was challenged with CiLV-C. In order to accomplish that, cDNA libraries were constructed from healthy and CiLV-inoculated sweet orange leaves. Two hundred and fifty-four genes were found to differ significantly in terms of expression, with 193 of them induced and 61 repressed after inoculation. Here we discuss the possible roles of a sub-set of these genes involved in metabolism, energy, signaling and cell rescue, defense and virulence, and indicate which kind of response may take place in the initial steps of the disease. Although the symptoms induced by CiLV-C in its compatible interaction with sweet orange resemble those of hypersensitive response (HR) in incompatible interactions, our data indicate that, apparently, the manifestation of leprosis symptoms should not be considered HR.

Phytophthora parasitica transcriptome, a new concept in the understanding of the citrus gummosis

Due to the economic importance of gummosis disease for the citriculture, studies on P. parasitica-Citrus interaction comprise a significant part in the Brazilian Citrus genome data bank (CitEST). Among them, two cDNA libraries constructed from two different growth conditions of the P. parasitica pathogen are included which has generated the PP/CitEST database (CitEST - Center APTA Citros Sylvio Moreira/IAC- Millennium Institute). Through this genomic approach and clustering analyses the following has been observed: out of a total of 13,285 available in the Phytophthora parasitica database, a group of 4,567 clusters was formed, comprising 2,649 singlets and 1,918 contigs. Out of a total of 4,567 possible genes, only 2,651 clusters were categorized; among them, only 4.3 percent shared sequence similarities with pathogenicity factors and defense. Some of these possible genes (103) corresponding to 421 ESTs, were characterized by phylogenetic analysis and discussed. A comparison made with the COGEME database has shown homology which may be part of an evolutionary pathogenicity pathway present in Phytophthora and also in other fungi. Many of the genes which were identified here, which may encode proteins associated to mechanisms of citrus gummosis pathogenicity, represent only one facet of the pathogen-host Phytophthora - Citrus interaction.