Gene Expression Profile During Apricot Fruit Growth, Using a Peach
Microarray
G.A. Manganaris, F. Ziliotto, A. Rasori, C. Bonghi and A. Ramina
Department of Environmental Agronomy and Crop Science
University of Padova
viale dell’ Università 16, 35020 Legnaro (Padova) Italy
R. Banfi, F. Geuna and D. Bassi Faculty of Agriculture
University of Milano
via Celoria 2, 20133 Milano Italy
P. Tonutti
Sant’Anna School of Advanced Studies Piazza Martiri della Libertà 33, 56127 Pisa Italy
Keywords: genomics tools, transcriptomics, oligonucleotide hybridization, fruit ripening,
Rosaceae, Prunus armeniaca, Prunus persica
Abstract
There is a high degree of sequence conservation within the Family Rosaceae and, in particular, among the Prunus species. The first available peach microarray (µPEACH1.0) was employed for the investigation of the transcription profile during apricot (Prunus armeniaca) fruit growth and development. Fruits (cv. ‘Goldrich’) were harvested at two growth stages, corresponding to immature (6 weeks before harvest) and ripe (at harvest) stage. Overall, this preliminary transcriptomic approach suggests that peach microarray can be employed in a heterologous fashion. Amongst the 746 genes which could be statistically analysed, 287 were induced, while 260 were down-regulated. In addition to the well documented role of genes implicated in ethylene biosynthesis perception and transduction as well as those involved in cell wall metabolism, this study brings into light a putative role for genes encoding auxin-regulated proteins, and those responsive to stress conditions and to other biotic or abiotic stimuli. The data of the present study can also be used for comparative purposes within Prunus species, since for 203 and 189 genes that were up- and down-regulated respectively during apricot fruit development, data exist for their regulation during the last stages of peach fruit ripening. Future experiments should include analysis of more fruit stages and data validation for the genes of interest.
INTRODUCTION
Transcript-profiling methods are increasingly used to understand the biological basis of growth and development, and in the case of fruits, of their quality. Such methods provide information for thousands of genes, including those of still unknown function. Furthermore, high-throughput methodologies can be used for comprehensive transcriptome analyses, which may lead to further elucidation of fruit growth and development up to the ripening stage (reviewed in Bonghi and Trainotti, 2006).
Microarray technology is suitable for large-scale trancriptomic analyses, through the simultaneous monitoring of many genes in a single experiment, avoiding limitations of traditional molecular techniques, while it can be utilized in a heterologous fashion for gene discovery in species where few resources are available, thus making it an attractive genomic tool. To date, a tomato microarray was successfully employed for comparative studies in Solanaceae (tomato, pepper, eggplant) (Moore et al., 2005).
Considering the high degree of sequence conservation within the Family Rosaceae and, in particular, among the Prunus species, the first available peach microarray
(µPEACH1.0) was applied to investigate changes in gene expression during apricot fruit development. This microaray contains 4,806 oligoprobes (70 bases long) each corresponding to a single unigene, as indicated in a dedicated database (ESTree consortium, 2005). This unigene collection comes from EST sequences mainly obtained from libraries of ripening fruits, so it is biased towards this physiological process (Trainotti et al., 2006).
MATERIAL AND METHODS
Fruit material (cv. ‘Goldrich’) was collected in two distinct stages: (1) green – immature stage (S1, 6 weeks before ripening), and (2) during the on-tree ripening (S2, firmness values ˜ 1 KgF). Such material was stored at -80°C until needed.
The RNA was isolated according to Bonghi et al. (1998). In order to remove contaminant DNA from the RNA samples, the nucleic-acid extract was treated with DNase, according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm and its integrity was checked on agarose gels. The conversion of RNA into target cDNA, microarray hybridization and data analysis were carried out as analytically described by Trainotti et al. (2007). Genes showing log2 ratio either > 1 (up-regulated) or < -1 (down-regulated) were annotated following the Gene Ontology categories (GO) developed by TAIR. In addition, functional annotation for the genes differentially transcribed was done using the VirtualPlant 0.9 software.
RESULTS AND DISCUSSION
The distribution of up-regulated (287) and down-regulated (260) genes during the transition of ‘Goldrich’ apricot fruit from ‘immature – green’ to ‘ripe – ready to eat’ stage, based on their homology to the Arabidopsis proteome is depicted in Fig. 1. The data of the present study can be also used for comparative purposes within Prunus species, since for 203 and 189 genes that were up and down-regulated respectively during apricot fruit development, data exist for their regulation during the last stages of peach fruit ripening (ESTree consortium, 2005, Trainotti et al., 2006). In that case, however, data should correspond to similar developmental stages of the fruit in order to dissect common and/or divergent mechanisms within the Prunoideae sub-family.
Among the 287 up-regulated transcribed genes, 48 were related to stimulus response. Among them, 22 were expressed as response to hormones and more particular to auxin (Fig. 2a). Members of the Aux/IAA family are highly up-regulated when ripening proceeds and their importance has been highlighted during the onset of peach fruit ripening (Trainotti et al., 2007). An up-regulation of genes encoding heat-shock proteins (HSP) was monitored (Fig. 2b), in accordance with data reported by Grimplet et al. (2005). Besides their well documented role as a response to stress conditions, HSP have been implicated in pectin depolymerization (Ramakrishna et al., 2003) and more recently were analysed in a proteomic level during tomato fruit development (Faurobert et al., 2007). In addition, a down-regulation of genes encoding for antioxidant enzymes (catalase, peroxidase, lipoxygenase, Fig. 2c) was monitored. A down-regulation of genes encoding for peroxidase and catalase has been also reported during the transition of apricot fruit from immature to mature stage (Grimplet et al., 2005). The regulation of other genes encoding some proteins of interest is depicted in Fig. 2.
Overall, results indicated that peach microarray can be successfully applied in related, yet phenotypically distinct, species belonging to the Sub-family Prunoideae. A thorough analysis of more fruit stages and data validation for the genes of interest should allow us to unravel the mechanisms underlying the ripening process in apricot and generally in stone fruits. Another future perspective should be the identification of genes differentially expressed during developmental stages of apricot fruit and their correlation with traits of agronomic interest.
ACKNOWLEDGEMENTS
G.A. Manganaris is a recipient of an E.U. Marie Curie individual fellowship (Grant MEIF-CT-2006-038997).
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0 2 0 4 0 6 0 8 0 1 0 0 cell wall othe r mem bran es chlo ropl ast othe r int race llula r com pone nt othe r cyt opla smic com pone nt nucle us plas tid mito chon dria cyto sol extra cellu lar ER Golg i app arat us plas ma mem bran e ribos ome unkn own cellu lar c ompo nent othe r cel lular com pone nts G O c e llu la r c o m p o n e n t (a ) u p -re g u la tio n d o w n -re g u la tio n 0 20 40 60 80 100 othe r enz yme activ ity unkn own mole cular func tion trans ferase acti vity hydr olase activi ty othe r bin ding prot ein b indin g kinas e ac tivity trans cript ion fa ctor a ctivi ty trans porte r act ivity nucle otide bind ing DNA or RNA bindin g nucle ic ac id bin ding othe r mole cular func tions struc tural m olecu le ac tivity rece ptor b indin g or a ctivi ty GO m olecular function (b) up-regulation down-regulation 0 2 0 4 0 6 0 8 0 1 0 0 othe r cell ular pro cess es othe r met aboli c pro cess es resp onse to a biot ic/bi otic stim ulus unkn own biolog ical p roce sses resp onse to s tress prot ein m etab olism othe r bio logica l pro cess es signa l tra nsdu ction trans port cell orga niza tion & bi ogen esis elect ron trans port/ ener gy p athw ay deve lopm enta l pro cess es trans crip tion DNA/ RNA met abol ism G O b io lo g ic a l p ro c e ss (c ) u p -re g u la tio n d o w n -re g u la tio n
Fig. 1. Distribution of up- and down-regulated genes during the transition of ‘Goldrich’ apricot fruit from ‘immature-green’ to ‘ripe’ stage, based on their homology to the Arabidopsis proteome, and grouped according to the terms used in the (a) Gene Ontology (GO) cellular component, (b) GO molecular function and (c) GO biological process by TAIR(The Arabidopsis Information Resource).
(a) 0 1 2 3 4 5 6 IAA 42 IAA 57 IAA 84 IAA 358 IAA 1020 IAA 1068 IAA 1741 IAA 1993 IAA 5160 TIR1 271 3 log 2 (S 2/S1 ) (b) 0 1 2 3 4 5 6 1-ACO 6 4 GcpE 407 HIP 2 25 HSP 306 5 HSP 306 7 HSP 3 072 HSP 346 2 log 2 (S 2 /S1 ) (c) -5 -4 -3 -2 -1 0 SAM synt 68 EREB F 75 0 EREB P 24 99 Deh ydrin 1528 Defens in116 2 PRP 452 4 CAT 10 24 POD 474 8 LOX 542 4 lo g2 (S2/S1)
Fig. 2. Modulation of gene expression, measured by means of the μPEACH1.0 microarray, during the transition of ‘Goldrich’ apricot fruit from ‘immature – green’ to ‘ripe’ stage. The genes analysed code (a) for proteins putatively involved in auxin metabolism, (b) in response to stress conditions with up-regulation effect and (c) in response to stimuli with down-up-regulation effect. Numbers after gene acronyms indicate the peach contig name. Bars correspond to ± SD of the means. IAA: auxin-induced or -regulated proteins, TIR1: transport inhibitor response 1-like protein, 1-ACO:1-aminocyclopropane-1-carboxylate oxidase, HIP:harpin-inducing protein, HSP:Heat-shock protein, SAM synt: S-Adenosylmethionine synthase, EREBF/P: ethylene-responsive element binding factor/protein, PRP:pathogenesis-related protein, CAT:catalase, POD:peroxidase, LOX:lipoxygenase.
