IICAR   25568
INSTITUTO DE INVESTIGACIONES EN CIENCIAS AGRARIAS DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The importance of being duplicated in the tomato sHSP gene family
Autor/es:
ARCE, DÉBORA; KRSTICEVIC, FLAVIA; TAPIA, ELIZABETH; CACCHIARELLI, PAOLO; PRATTA, GUILLERMO R.; SPETALE, FLAVIO; PONCE, SERGIO
Lugar:
Santiago de Cali
Reunión:
Congreso; IV Congreso Colombiano de Bioinformática y Biología Computacional y la VIII Conferencia Iberoamericana de Bioinformática; 2017
Institución organizadora:
Sociedad Colombiana de Bioinformática y Biología Computacional SC2B2 y la Sociedad Iberoamericana de Bioinformática SoIBio
Resumen:
ABSTRACTIn living organisms, small heat shock proteins (sHSPs) are triggered in response to stress situations.Evolutionary relationships in sHSP genes have been investigated in rice, potato and tomato genomeshighlighting the importance of tandem duplication events for the expansion of sHSP gene family in plants.Additionally, transcriptomic studies have revealed complex patterns of expression for these genes duringfruit ripening. This work aims to shed light on the way the expression patterns of sHSPs genes getaffected by variations in their nearby promoter architecture by taking advantage of their redundantpresence. HSEs indeed appeared in the regulatory region of most up-regulated sHSPs genes duringtomato fruit ripening, however some striking exceptions are observed. Conversely, regulatory regions ofdown-regulated, not-differentially expressed or not-expressed sHSP genes are almost depleted of HSEs.Concerning the exceptions, we note Solyc09g015000, arising from tandem duplication event inchromosome 9. This uncharacterized sHSP gene is strongly up-regulated during fruit ripening (and heatshock) but is completely depleted from HSEs. Instead, a unique short CTAGA non-HSE motif, resemblinga degenerated HSE sequence, is present as confirmed by similar results in orthologous promoters fromS. tuberosum and ​ S. pennelli ​ . Preliminary results from predicted protein-protein interactions involving thesHSP protein family and heat shock response (HSR) key factors (HSP70s, HSP90 and MBF1c) suggestthat Solyc09g015000 could be mediating the stress response through a regulatory network of the ringtype involving four sHSP proteins, two of them of tandem origin, and one uncharacterized protein with anATPase domain typical of the HSP100 family.RESULTSAiming at a precise characterization of HSEs in the promoter region of tandem duplicated sHSP genes intomato, a position weight matrix (PWM) approach is considered. For this purpose, a careful data-curationprocess over the gene expression data reported in [1] was performed. This curation process includesselection of best suitable algorithms [2]?[4] for PWM modeling of HSEs, together with stringent PWMquality controls as indicated [5] (Fig. 1A). Resulting PWMs were used to scan the promoter region of the33 members in the sHSP gene family of ​ S. lycopersicum (cv Heinz 1706) with main focus on the fivephysical clusters of tandem duplicated sHSPs genes. Additionally, PPIs analysis for predictingprotein-protein interactions in sets of co-regulated genes during tomato fruit ripening is considered. Jointlyanalyses of these results contribute to improve the sparse annotation of the sHSP gene family andinteractors in tomato.Cis-acting elements in the regulatory regions of sHSP genes related to the ripening processGene promoter architecture characterization of sHSP gene family in ​ S. lycopersicum ​ cv Heinz 1706 canshed light on its functional annotation. A pattern-oriented screening of transcription factor binding sites(TFBS) families against the regulatory region of sHSP genes showed the over representation of HSE,ERE, ABA and WRKY TFBS families. To avoid false positives, only over represented TFBS of minimumsize 5 nt were considered. As a result, only the HSE, ERE and ABA TFBS families remained fordownstream statistical analysis with QC PWMs (p-value=1E-04) (Fig. 1A) in the regulatory region of the33 members of sHSP gene family. HSEs indeed appear in the regulatory region of most sHSPsup-regulated genes during tomato fruit ripening [6]?[8] (Fig. 1B). Conversely, regulatory regions ofdown-regulated, not-differentially expressed or not-expressed sHSP genes during fruit ripening werealmost depleted of HSEs. This trend on HSEs, increasing their presence for up-regulated members whiledepleting it for the others, holds also tandem duplicated sHSP genes (Fig. 1B). Disentangling the actualeffect of the HSE presence in tandem duplicated genes may help to better understand the functionality ofubiquitous sHSP genes. In this regard, HSEs are observed for almost all up-regulated tandem duplicatedsHSP genes. In particular, two physical clusters of sHSP genes, one located in chromosome 6(Solyc06g076520, Solyc06g076540, Solyc06g076560 and Solyc06g076570) and the other inchromosome 8 (Solyc08g062340 and Solyc08g062450) (Fig.1B, c and d), show the HSE presence. Also,HSEs heavily populates the unique up-regulated member (Solyc08g078700) in the remaining physicalcluster of three sHSP genes located in chromosome 8. For the other two nearby members of this cluster,HSEs are just present (Solyc08g078720) or absent (Solyc08g078710), consistently with their observedexpression profiles (Fig.1B e). An exception to the apparent rule of HSE presence and sHSPup-regulation is observed in the physical cluster of two sHSP genes in chromosome 9. In this case, bothsHSP genes are up-regulated but just one of them (Solyc09g015020) shows the HSE presence while theother (Solyc09g015000) seems to be completely depleted from HSEs. (Fig.1B f). Finally, Fig. 1B (b)shows a heterogeneous expression pattern of tandem duplicated sHSPs genes in a phylogenetic cluster(not a physical one) with an expected HSE presence.PPIs analysisHSE presence exception to sHSP upregulation during stress, including fruit ripening, rises the questionabout its actual importance for triggering sHSP expression. In this regard, the study of PPIs may shedlight on this issue. We wonder if Solyc09g015000 may be behave like MBF1, known to be a key factorduring thermoregulation in Arabidopsis despite the HSE absence in its promoter region [9], PPI analysisfrom up-regulated genes during fruit ripening and HS was conducted (Fig. 2A and C). Up-regulated genesinclude those in the HSP90 and HSP70 families known to contribute in maintaining cellular homeostasisduring stress conditions in human, Arabidopsis and tomato [10], [11], [12]. HSP90 constitutesapproximately 1-2 % of the total protein content in eukaryotes suggesting complex interconnections withother key regulators and cochaperones in response to stress [12]. Moreover, HSP70 and HSP100participate with HSP90 during the proteome response to stress also contributing to maintain cellularhomeostasis in physiological and stress conditions [13]. Despite small variations in their order ofappearance, HSP90, HSP70 and HSP100 proteins are the first barrier or fence in response to stressconditions in different organisms. This observation motivates a computational strategy for uncovering thefunctionality of uncharacterized sHSPs in tomato, including the striking Solyc09g015000. When removingcharacterized HSP90, HSP70 and HSP100 proteins, a ring network with the same five nodes emerged inboth ripening and HS (Fig. 2B and D, respectively). In this scenario, Solyc09g015000 mediates betweenannotated Solyc03g082420, belonging to the ring, and the well characterized sHSP Solyc05g014280,outside the ring. GO MF and CC computational analysis of Solyc03g082420 and Solyc05g014280suggests that Solyc09g015000 is a cytosolic protein probably which binds to ribosomes or RNAcontributing to the first barrier or fence in response to stress.MAIN CONTRIBUTIONIn silico promoter analysis of the sparsely characterized sHSP gene family in tomato was performed.Quality controlled PWMs were estimated from curated transcriptome and microarray datasets. PWMsrevealed unusual promoter architectures in certain uncharacterized tandem duplicated sHSPs largelyreported to be associated with fruit ripening. Aiming to disentangle the functionality of these genes, weadvance into the study of PPIs during different type of stresses. To uncover the actual functionality oftarget sHSP genes, PPI networks were analyzed in terms of stress pathways. Hence, well-knownupstream, first-barrier, HSPs involved in stress responses, including HSP 100s?, HSP90s? and HSP70s?,were removed from PPI networks. As a result, background PPI networks involving just sHSPs andcomplimentary uncharacterized proteins were obtained. Despite the stress type, a striking ringarrangement involving four sHSPs and one HSP100-like protein emerged. Among the four sHSPs, two ofthem came from tandem duplication events. These newly discovered subnetworks, mostly built fromsHSPs, may serve as backup stress response networks once upstream HSPs (90-70) have beendefeated.REFERENCES[1] S. Fragkostefanakis, S. Simm, P. Paul, D. Bublak, K. D. Scharf, and E. Schleiff, ?Chaperone network composition inSolanum lycopersicum explored by transcriptome profiling and microarray meta-analysis,? ​ Plant, Cell Environ. , ​ vol. 38, no.4, pp. 693?709, 2015.[2] S. Luehr, H. Hartmann, and J. 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Tapia, ?Tandem duplication events in the expansion of the small heat shockprotein gene family in Solanum,? ​ G3 Genes Genomes Genet. , ​ vol. X, no. August, 2016.[7] K. Scharf, M. Siddique, and E. Vierling, ?The expanding family of Arabidopsis thaliana small heat stress proteins and a newfamily of proteins containing alpha -crystallin domains ( Acd proteins ),? ​ Cell Stress Chaperones , ​ vol. 6, pp. 225?237, 2001.[8] M. H. Al-Whaibi, ?Plant heat-shock proteins: A mini review,? ​ J. King Saud Univ. - Sci. ​ , vol. 23, no. 2, pp. 139?150, 2011.[9] Suzuki, Nobuhiro et al. ?Identification of the MBF1 Heat-Response Regulon of Arabidopsis Thaliana.? ​ The Plant journal : forcell and molecular biology. v ​ ol. 66, no.5, pp. 844?851. Jun. 2011.[10] P. Barah, N. Jayavelu, R. Sowdhamini, K. Shameer, and A. Bones, ?Transcriptional regulatory networks in Arabidopsisthaliana during single and combined stresses.,? ​ Nucleic Acids Res. , ​ vol. 44, no. 7, pp. 3147?3164, 2016.[11] P. Jacob, H. Hirt, and B. Abdelhafid, ?The heat-shock protein/chaperone network and multiple stress resistance,? ​ PlantBiotechnol. J. , ​ vol. 15, no. 4, pp. 405?414, 2017.[12] K. Krukenberg, T. Street, L. Lavery, and D. Agard, ?Conformational dynamics of the molecular chaperone hsp90.,? ​ Q. Rev.Biophys , ​ vol. 44, no. 2, pp. 229?255, 2011.[13] A. Mogk, E. Kummer, and B. Bukau, ?Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation,?Front. Mol. Biosci. ​ , vol. 2, no. May, pp. 1?10, 2015.Figure 1. ​ HSEs in the regulatory regions of ​ S. lycopersicum cv Heinz 1706 tandem duplicated sHSP genes related to theripening process. A) PWM logos (left panels) and PWMs quality controls using RSAT matrix-distrib (middle graphs) andmatrix-quality (right graphs) for HSE (HSF21 and HSF1AE). B) Matrix-scan in tomato tandem duplicated sHSPs gene regulatoryregions. Differential gene expression profiles are indicated as up-regulated (UP, red triangles), down-regulated (DOWN, greentriangles), not differentially expressed (NDE) and absence of expression (NE) in red compared to green fruits during ripening.Figure 2. ​ Predicted protein-protein interactions (PPIs) analysis during ripening and heat stress in tomato​ . ​ Interactome of theripening (24 genes, A) and heat stress (23 genes) named 5t and 10s clusters, showing 24 and 22 nodes and with 36 and 65 edges,respectively in the protein-protein interaction maps. Genes were denoted as nodes in the graph and interactions between them werepresented as edges. Two subnetworks (B and C) were constructed using conservative combined score threshold of 0.5 (mediumlevel) considering six channels of evidence (Experiments, Database, Textmining, Coexpression, Fusion and Co-occurrence) for 5tand 10s clusters. Nodes and links represent tomato (​ S. lycopersicum ​ cv Heinz 1706) proteins and protein interactions, respectively.