Industry

Berry skin development in Norton grape: Distinct patterns of transcriptional regulation and flavonoid biosynthesis

Description
Berry skin development in Norton grape: Distinct patterns of transcriptional regulation and flavonoid biosynthesis
Categories
Published
of 23
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  RESEARCH ARTICLE Open Access Berry skin development in Norton grape: Distinctpatterns of transcriptional regulation andflavonoid biosynthesis Mohammad B Ali 1,4 , Susanne Howard 1 , Shangwu Chen 3 , Yechun Wang 2 , Oliver Yu 2 , Laszlo G Kovacs 1 ,Wenping Qiu 1* Abstract Background:  The complex and dynamic changes during grape berry development have been studied in  Vitisvinifera , but little is known about these processes in other  Vitis  species. The grape variety  ‘ Norton ’ , with a majorportion of its genome derived from  Vitis aestivalis , maintains high levels of malic acid and phenolic acids in theripening berries in comparison with  V. vinifera  varieties such as Cabernet Sauvignon. Furthermore, Norton berriesdevelop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remainsusceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit acomprehensive analysis of transcriptional regulation and metabolite pathways. Results:  A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to fullripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that atotal of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between atleast two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes duringberry development. Transcript levels of   PR-1  in Norton berry skin clearly increased during the ripening phase. As inother grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patternsof six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyaninsand proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and CabernetSauvignon. Transcriptional patterns of   MYB5A  and  MYB5B  were similar during berry development between the twovarieties, but those of   MYBPA1  and  MYBPA2  were strikingly different, demonstrating that the general flavonoidpathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3 ’ -O-hydroxylase ( F3 ’ H  ), flavonoid-3 ’ ,5 ’ -hydroxylase ( F3 ’   5 ’   H  ), leucoanthocyanidindioxygenase  (LDOX) , UDP-glucose:flavonoid 3 ’ -O-glucosyltransferase ( UFGT  ), anthocyanidin reductase (  ANR ),leucoanthocyanidin reductase ( LAR )  1  and  LAR2  in berry skin of Norton than in those of Cabernet Sauvignon. It wasalso found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignonberry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulationpatterns in Norton berry skin. Conclusions:  This study provides an overview of the transcriptome changes and the flavonoid profiles in the berryskin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of   PR-1  and stilbene synthase genes likely contributes to the developmentally regulated resistanceduring ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase * Correspondence: WenpingQiu@missouristate.edu 1 Center for Grapevine Biotechnology, William H. Darr School of Agriculture,Missouri State University, Mountain Grove, MO 65711, USAFull list of author information is available at the end of the article Ali  et al  .  BMC Plant Biology   2011,  11 :7http://www.biomedcentral.com/1471-2229/11/7 © 2011 Ali et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the srcinal work is properly cited.  gene in berry development and disease resistance. Transcriptional regulation of   MYBA1 ,  MYBA2 ,  MYB5A  and  MYBPA1 as well as expression levels of their putative targets  F3 ’ H, F3 ’ 5 ’ H, LDOX  ,  UFGT  ,  ANR, LAR1 , and  LAR2  are highlycorrelated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a uniquepattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enologicalcharacteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties. Background Berry development in grapes is a complex process of physiological and biochemical changes [1]. It is initiatedby hormonal signals generated after pollination [2]. Thenature and srcin of the hormonal signals that influencethe complex processes of berry development have notbeen fully understood, but abscisic acid, brassinosteroidsand ethylene have been implicated in these processes[3,4]. Although ethylene is present at the beginning of  ripening, it does not show a rapid increase in concentra-tion, and no burst of respiration occurs in grape berries[5]. Thus, grapes are non-climacteric fruits.The berry development of grape follows a double-sigmoid pattern that is characterized by two growthphases interrupted by a lag phase (véraison) whichmarks the transition from herbaceous development toripening [6]. High-throughput profiling of transcripts by using the first generation Affymetrix Vitis GeneChip hasprovided a comprehensive picture of gene regulationthat depicts the complex biochemical pathways duringberry development of   V. vinifera  grapevines [7,8]. The transcriptome analysis has also identified distinct tran-scriptional patterns and tissue-specific genes in seed,skin and pulp of grape berry [9]. The results of thesestudies have offered the insights into how key regulatory circuits orchestrate berry development and influenceunique berry characteristics in  V. vinifera  varieties.The skin of grape berries serves as a physical and bio-chemical barrier that protects ripening berries frombeing attacked by pathogens. During the first growthphase, the skin accumulates high levels of proanthocya-nidins (PAs). The astringent properties of PAs may play a role in repelling herbivores from consuming berriesbefore seeds are mature, and also in the protection of plants against fungal pathogens [10]. At véraison, theskin begins to accumulate anthocyanins which are thepredominant pigments of grape berries. The dark coloris believed to attract herbivorous animals to promotethe dissemination of seeds into new territories. Support-ing this proposition is the fact that the skin color of wild  Vitis  species berries is black. In addition to PAsand anthocyanins, the skin also accumulates flavan-3-olmonomers, although the majority of flavan-3-ols aresynthesized in the grape seed [11]. The endo- and meso-carp of the berry contain large quantities of acids,primarily malic and tartaric acids, during the firstgrowth phase, and sugars during the second growthphase of berry development [1,2]. Prior to maturity, the skin ’ s resistance against patho-gens increases in order to protect the ripening grape ber-ries [12-14]. The high levels of flavonoid compounds in the skin are thought to contribute to the enhanced dis-ease resistance of mature berries. It was discovered thatmany highly expressed genes in the skin of CabernetSauvignon are associated with pathogen resistance andflavonoid biosynthesis [9]. The transcriptional profiles of skin-specific genes, which were also corroborated by pro-teomics analysis, indicated that a set of enzymes in theanthocyanin biosynthesis pathway were significantly over-expressed in the skin of fully ripe berries [15]. A setof pathogenesis-related (  PR ) genes, such as  PR-1, PR-2, PR-3, PR-4   and  PR-5  , all increased in the ripening berry of Cabernet Sauvignon, with  PR-3  and  PR-5   having themost dramatic increase [7,16]. During véraison, the berry  experiences a burst of reactive oxygen species (ROS) anda surge in the expression of genes that encode enzymesinvolved in the generation of antioxidants [8]. Generationof ROS is closely associated with cell death and plantdefense responses [17]. The timing of accumulation of these defense-related proteins is synchronized with theinitiation of the ripening berry  ’ s ability to prevent infec-tion by pathogens [18]. There is experimental evidencethat the increased expression of defense-related genesforms a protective layer in the berry skin against patho-gens [19,15]. This supports the hypothesis that there is a correlation between the increased expression of defense-related genes and the enhanced resistance against patho-gens in the ripening berry.The composition, conjugation and quantity of antho-cyanins in red varieties determine the color density andhue of the berry skin. Anthocyanins and PAs contributeto the astringency of wine and are also antioxidants withbeneficial effects on human health [20]. Transcriptionalregulation of the flavonoid pathway genes has been inves-tigated mostly in  V. vinifera  varieties. Six MYB transcrip-tion factors (MYBA1, MYBA2, MYB5A, MYB5B,MYBPA1 and MYBPA2) are associated with the regula-tion of the structural genes in the flavonoid pathway.MYBA1 and MYBA2 play roles in the biosynthesis of anthocyanins by activating the promoter of   UFGT  Ali  et al  .  BMC Plant Biology   2011,  11 :7http://www.biomedcentral.com/1471-2229/11/7Page 2 of 23  [21-23], which catalyzes the last step of anthocyanin synthesis. MYB5A and MYB5B are involved in regulatingseveral flavonoid biosynthesis steps [24]. MYBPA1andMYBPA2 regulate the last steps of pathways in the pro-duction of PAs [22,25]. Norton is considered a  V. aestivalis- derived variety which produces high quality red wine that is comparableto wines made from  V. vinifera  grapes. Norton leavesaccumulate high levels of salicylic acid (SA) and SA-associated defense genes in comparison with CabernetSauvignon. Abundant SA and high expression of SA-associated defense genes may equip Norton grape with arobust innate defense system against pathogens [26].Furthermore, total amounts of anthocyanin and phenolicacid contents are significantly higher in Norton berriesthan in those of   V. vinifera  [27,28]. Similarly to other grape varieties that srcinate in North America, Nortonberries develop exceptionally high levels of disease resis-tance, which enable viticulturists to grow this grape withminimal application of pesticides in regions with highdisease pressure. Transcriptomics, proteomics, andmetabolic profiles of berry development of   V. vinifera  varieties Cabernet Sauvignon and Pinot Noir have beenstudied and documented using Affymetrix GeneChips[7,8,15,29]. Consequently, the synthesis of flavonoids in the berry skin, and the expression and regulation of theunderlying genes are well understood in  V. vinifera . Lit-tle is known, however, about the regulation of the bio-synthesis of flavonoid compounds in the berry skin of Norton. In this study, we analyzed the transcriptionalprofiles of over twenty thousand genes in Norton berry skin across six developmental stages using the secondgeneration of Affymetrix  Vitis  microarrays (GRAPEGENGenChip) [30]. We discovered a high coordinationbetween the transcriptional regulation of key transcrip-tion factors and structural genes in the flavonoid bio-synthesis pathway and the accumulation profiles of flavonoid compounds. Comparative analysis of key genes in flavonoid biosynthesis and of the main flavo-noid compounds between Norton and Cabernet Sau- vignon revealed variety-specific patterns of generegulation and compound biosynthesis. The results fromthis study yield new knowledge on the distinct chemistry and characteristics of Norton grapes. Results and Discussion Discovery of differentially expressed genes during Nortonberry skin development Similarly to the berry development of   V. vinifera  vari-eties, the development of Norton berries is characterizedby a two-stage growth pattern. Sugar accumulationbegan at the early stages and accelerated during vérai-son. Also following the pattern of   V. vinifera  berry development, the levels of titratable acidity dropped atstage 34 (at 66 days after bloom [DAB]) and continuedto decrease until the berry was ripe. The descriptors of berry development, including berry diameter, titratableacidity and soluble solids, are presented in an accompa-nying paper (Ali  et al. , in preparation). We started sam-pling on June 26, 2008 when the skin could beseparated from the pulp of the berry. At this point, theberry was at stage 31 (17 DAB) on the Eichorn-Lorenzphenological scale. Subsequently, skin samples weretaken at stages 33, 34, 35, 36, 37 and 38, correspondingto 37, 66, 71 (véraison), 85, 99, and 127 DAB. Skin tis-sue was frozen in liquid nitrogen and total RNA wasextracted subsequently. The RNA was then labeled andhybridized to GRAPEGEN Affymetrix GeneChips. Pro-cessing of raw intensity values in CEL files and subse-quent normalization and Median polishing weredescribed in the paper (Ali  et al  ., in preparation).A Principal Component Analysis (PCA) of the eigh-teen arrays was performed to assess the similarity of expression values among the replicates (AdditionalFile 1). The results from the PCA indicated a highdegree of similarity among three biological replicatesthat were clustered tightly within the scatterplot. Inaddition, PCA showed that data of two proximal devel-opmental stages were more similar to each other thandata of distal developmental stages. There is a clearalignment and separation of developmental stages alongthe PC1 in the plot (Additional File 1). The eighteensets of the data were then converted to z-scores andsubjected to two-way unsupervised agglomerative clusteranalysis (Additional File 2). This analysis showed thateach stage represents a major branch which containsonly the three biological replicate data for that stage.The results from these two analyses demonstrated thatthere is a good reproducibility among the three biologi-cal replicates and thus all data were included in the ana-lysis. Pearson correlation coefficients between biologicalreplicates were also calculated and were in the range of 0.9812 to 0.9976 (Additional File 3), further corroborat-ing significant correlations between biological replicatesin each developmental stage.After the data of all eighteen arrays were processed andassessed for quality, the error-weighted intensity experi-ment definitions (EDs) were calculated by averaging theintensity of three biological replicates for each stage andthen error-corrected using the Rosetta error model [31].ANOVA was conducted on the error-weighted intensity of three biological replicates at each stage across sixdevelopmental stages with the Benjamini-Hochberg FalseDiscovery Rate multiple test correction [32]. This resultedin the discovery of 15,823 probe sets that exhibited signif-icant variations at the transcript levels between at leasttwo developmental stages at  P   ≤  0.001 (Additional File 4).The differentially expressed probe sets comprise more Ali  et al  .  BMC Plant Biology   2011,  11 :7http://www.biomedcentral.com/1471-2229/11/7Page 3 of 23  than 78% of all probe sets on the microarray, indicatingthat a large number of genes represented on the array changed significantly at transcript levels at some pointsduring berry development. To discover the genes whosetranscript levels varied significantly from a baseline calcu-lated from all six developmental stages, the intensity EDsof each probe set were divided by an error-weighted aver-age of all six developmental stages. Under the criteria of absolute fold-change  ≥ 2.0 in at least one developmentalstage and having a LogRatio  P   -value  ≤  0.001 in at leastone stage, we identified 3,352 probe sets (Additional File5). We selected this group of the most significantly expressed genes for the subsequent analysis. The largenumber of transcripts that changed at expression levelscorroborated earlier findings that genes of different func-tions were detected in the berry skin at the beginning of  véraison and the later stages of ripening, reflecting thedramatic biochemical changes that take place duringberry ripening [7,15]. Cluster analysis of differentially expressed genes inNorton berry skin We used the nucleotide sequence from which each setof probes was designed to acquire the best-matchedGSVIVT ID in Genoscope (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/) or TC number in DFCIGrape Gene Index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=grape). The total of 3,352probe sets represented 2,760 unique genes. We removedthose probe sets where more than one probe set wasassigned to the same GSVIVT ID or TC numbers butshowed different expression patterns, and compiledthem into a separate file for future analysis. At thistime, it is not possible to discern what factors, such asalternatively spliced transcripts or degradation biases of the 5 ’ -end and 3 ’ -end portion of mRNA, influence theexpression levels of these genes. We subjected the Log 2 -transformed fold-change of the remaining 2,359 uni-genes to clustering by the  k  -means method. A total of 20 clusters were defined from this group of genes basedon the figure of merit value (Additional File 6).Transcript abundance of these genes in cluster 1, 12,13, 18 and 20 increased after véraison (Figure 1). Thesefive clusters contained a total of 1,053 genes. Cluster 11(113 genes) and Cluster 16 (42 genes) represented a pat-tern of transient increase and decrease, respectively, of transcript levels at the onset of véraison and subse-quently unchanged post-véraison. The expression pat-tern of cluster 8 (65 genes) and cluster 19 (60 genes)was reciprocal. In cluster 8, transcript levels increasedpre-véraison and decreased post-véraison. In cluster 19,transcript levels decreased at véraison, but increasedboth pre-véraison and post-véraison. The remaining ele- ven clusters included 1,026 genes and exhibited apattern of steady decline post-véraison. The genes ineach cluster are listed in Additional File 6. Developmental regulation of defense-related genes A total of 48 differentially expressed genes were asso-ciated with defense, disease resistance, and hypersensi-tive response (Table 1). Among them, twenty onegenes were up-regulated, and twenty five genes weredown-regulated post-véraison. These defense-relatedgenes include the well characterized polygalacturonaseinhibiting protein (PGIP), dirigent protein, NBS-LRR,Non-race-specific disease resistance 1 (NDR1), pow-dery mildew resistant 5 (PMR5), and harpin-inducedprotein 1 genes.Especially noticeable is the expression profile of the  PR-1  gene, which is an indicator for the inductionof local defense and systemic acquired resistance(SAR) in plants [33,34]. In grapevine, the  PR-1  gene(GSVIVT00038581001) was induced by salicylic acid[35], and up-regulated after infection with the powdery mildew (PM) fungal pathogen  Erysiphe necator   [26].Transcript levels of   PR-1  increased progressively post- véraison in both Norton (cluster 18, Figure 1 andTable 1), and Cabernet Sauvignon [7,29]. The gene  AtWRKY75   plays an important role in the activation of basal and resistance (  R ) gene-mediated resistance inArabidopsis [36], and transcript levels of its grapevineortholog increased in response to PM infection [26].Interestingly, the grapevine  WRKY75   ortholog was dis-covered in cluster 18. Four NBS-LRR genes were alsoidentified in cluster 18, indicating these proteins areregulated developmentally in grape (Table 1). PlantNBS-LRR proteins are receptors that directly or indir-ectly recognize pathogen-deployed proteins, and thisspecific recognition triggers plant defense responses[37,38]. In some cases, they also play a role in the regu- lation of developmental pathways [39].Five probe sets were annotated as thaumatin-like pro-teins and two as osmotins. Their transcript levelsincreased significantly in the late stages of Norton berry development (Additional File 5 and 6), as was shown previously in varieties of   V. vinifera  [7,29]. Thaumatin- like proteins inhibit spore germination and hyphalgrowth of   E. necator  ,  Phomopsis viticola , and  Botrytiscinerea  [40]. We found that transcript levels of five chit-inase genes increased post-véraison in Norton berry skin(cluster 12, 13, 19, and 20). Transcript levels of basicclass I (VCHIT1b) and a class III (VCH3) chitinase of grapevines increase in response to the chemical activa-tors of SAR and are considered as markers of SAR [41].Furthermore, enzymatic activities of chitinase and ß-1,3-glucanase also increase during berry development in theabsence of pathogens [15]. Non-specific lipid transferproteins (nsLTPs) belong to a family of small cystein-rich Ali  et al  .  BMC Plant Biology   2011,  11 :7http://www.biomedcentral.com/1471-2229/11/7Page 4 of 23  Figure 1  Clustering of the expression profiles of 2,359 genes that were defined as significantly changed across the six developmentalstages of Norton berry skin . Clustering was performed using  k  -means statistics and 20 clusters were chosen for further analysis of transcriptional patterns. The number of genes in each cluster is listed in parenthesis. The X-axis indicates grape berry developmental stages indays after bloom (DAB); The Y-axis indicates the Log 2 -transformed fold-change of stage-specific intensity relative to the baseline intensity of eachgene. The véraison phase is denoted by purple bar. A list of genes, their ChipID, Genoscope ID, putative function, Enzyme ID and pathway inVitisnet for each cluster is included in Additional File 6. Ali  et al  .  BMC Plant Biology   2011,  11 :7http://www.biomedcentral.com/1471-2229/11/7Page 5 of 23
Search
Similar documents
View more...
Tags
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x