Global gene expression analysis of apple fruit development from the floral bud to ripe fruit

1471-2229-8-16 1471-2229 Research article Global gene expression analysis of apple fruit development from the floral bud to ripe fruit Janssen J Bart bjanssen@hortresearch.co.nz Thodey Kate Kate.Thodey@bbsrc.ac.uk Schaffer J Robert RSchaffer@hortresearch.co.nz Alba Rob rma28@cornell.edu Balakrishnan Lena lena.b@xtra.co.nz Bishop Rebecca becklesbishop@hotmail.com Bowen H Judith jbowen@hortresearch.co.nz Crowhurst N Ross rcrowhurst@hortresearch.co.nz Gleave P Andrew AGleave@hortresearch.co.nz Ledger Susan SLedger@hortresearch.co.nz McArtney Steve Steve_McArtney@ncsu.edu Pichler B Franz f.pichler@auckland.ac.nz Snowden C Kimberley KSnowden@hortresearch.co.nz Ward Shayna sward@hortresearch.co.nz

The Horticulture and Food Research Institute of New Zealand Ltd., Mt Albert, Private Bag 92169, Auckland Mail Centre, Auckland 1142, New Zealand

John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

22 Ramphal Terrace, Khandallah, Wellington, New Zealand

4 La Trobe Track, RD2 New Lynn, Karekare, Auckland, New Zealand

Department of Horticultural Science, North Carolina State University, Mountain Horticultural Crops Research and Extension Centre, 455 Research Drive, Fletcher, NC 28732-9244, USA

Microbial Ecology & Genomics Lab, School of Biological Sciences, University of Auckland, Auckland, New Zealand

Monsanto Company – O3D, Product Safety Center, 800 North Lindbergh Blvd., St. Louis, MO 63167, USA

BMC Plant Biology 1471-2229 2008 8 1 16 http://www.biomedcentral.com/1471-2229/8/16 18279528 10.1186/1471-2229-8-16 13 9 2007 17 2 2008 17 2 2008 2008 Janssen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45–55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple.

Results

Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes.

Conclusion

Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Background

Fruit-bearing crop species are an important component of the human diet providing nutrition, dietary diversity and pleasure. Fruit are typically considered an enlarged organ that surrounds the developing seeds of a plant, or the ripened ovary of a flower together with any associated accessory parts 1. The development and final form of the fruiting body is widely varied, ranging from minimally expanded simple dehiscent (non-fleshy) fruit of the model plant Arabidopsis, through expanded ovaries of tomato, to complex fruiting organs with several different expanded tissues, such as found in the pome fruit 1. Common to all fruit is the developmental process that results in expansion of tissue near the seed in a coordinated manner with seed development (usually, but not always, enclosing the seed). At early stages during development (both before and after successful fertilization, and sometimes in the absence of fertilization) the fruit tissue undergoes several rounds of cell division, followed (usually) by cell expansion during which the fruit stores metabolites and energy, in the form of starch or sugars (e.g. tomato development 234). Subsequently, usually after the seeds mature, the fruit undergoes a series of biochemical changes that convert starches into more available and attractive compounds, such as sugars, as well as producing volatile secondary metabolites that are thought to function as attractants for animals or insects which disperse the seed.

Morphological and physiological studies of fruit have led to considerable understanding of the physical and biochemical events that occur as fruit mature and ripen 135, however it is only relatively recently that genomic approaches have been used to investigate fruit development 46789. As a result of excellent genetic resources and the application of molecular and genomic approaches, tomato has become the best studied indehiscent fruit. Domestication of tomatoes has resulted in the increase of fruit size from a few grams to varieties 1000-fold larger 10. The physiological events leading to the expansion of the ovary wall of the tomato flower and in particular the events that occur around tomato ripening have been well described (for reviews see Gillaspy et al. 2; Giovanonni 3). More recently, molecular approaches have been used to study global gene expression in tomato 111213 allowing identification of large numbers of genes potentially involved in fruit development and ripening.

In other fruit crops, microarrays have been used to examine gene expression during the development and in particular the ripening of fruits such as strawberry 6, peach 14, pear 15, and grape 89. These studies have identified genes involved in fruit flavour and genes associated with distinct stages of fruit development.

Apples (Malus × domestica Borkh. also known as M. pumila) are members of the Rosaceae family, sub family pomoideae, which includes crop species such as pear, rose and quince. Members of the pomoideae have a fruit that consists of two distinct parts: an expanded ovary corresponding to the "core" which is homologous to the tomato fruit; and the cortex or edible portion of the fruit which is derived from the fused base of stamens, petals and sepals 116, which expands to surround the ovary. Fruit develop over a period of 150 days from pollination to full tree ripeness with a simple sigmoidal growth curve 1718. Physiological studies of apple fruit development have focused on measures of ripeness such as colour changes and breakdown of starch to form the palatable sugars. From such studies, it has been shown that floral buds contain a small amount of starch that is metabolized quickly after pollination. Starch levels then build up in fruit coordinate with cell expansion. At about 100 days after pollination starch levels begin to decline again and fruit sugars increase, until the fruit are fully ripe 19. Like tomato, apple undergoes an ethylene-dependent ripening stage 2021 and transgenic apples with reduced ethylene production fail to produce skin colour changes and appear to lack production of volatile compounds typically associated with apples 22.

Apple is functionally a diploid with 2n = 34 and a genome of moderate size (1C = 2.25 pg 23 which corresponds to approximately 1.5 × 10⁹bp) making genomic approaches to the study of its biology reasonable. Recently an EST sequencing approach has been used to identify apple genes 24; unigenes derived from this sequencing project were used to design the oligonucleotides used in this work. Two groups have published apple microarray analyses 2225. Lee et al. 25 used a 3484 feature cDNA array to identify 192 apple cDNAs for which expression changes during early fruit development. Using the same ~13000 gene (15726 feature) apple oligonucleotide array described in this paper, Schaffer et al. 22 identified 944 genes in fruit that respond to ethylene treatment and associated changes in gene expression with changes in fruit volatiles.

In the work described in this paper, microarrays have been used to study the developmental processes occurring during fruit formation from pollination to full tree ripeness. In pome fruit both core (ovary) and cortex (hypanthium) tissues expand. Understanding the regulation of the events required to produce a complex apple fruit, including the division and expansion of cells from different floral structures is the ultimate aim of this work. Using microarrays we show that large groups of genes are co-ordinately expressed at specific stages of fruit development. We have identified cell division genes for which expression coincides with the period of cell division in apple fruit and have identified starch metabolic enzymes likely to be involved as fruit store and then metabolize starch. Using a comparative approach we have identified a number of genes for which expression patterns are similar in both apple and tomato fruit development and may be involved in similar fundamental processes in fruit development.

Results

Microarray analysis of apple fruit development

When apple trees (Malus domestica 'Royal Gala') were at full bloom (greater than 50% of buds open) individual fully open flowers were tagged and trees separated into two biological replicates (Rep1 and Rep2). Based on physiological and morphological studies of apple fruit development 1719 eight time points were selected for sampling (Figure 1). The first sample 0 Days After Anthesis (DAA) was taken at the same time that fully open flowers were tagged. The 14 and 25 DAA sampling time points coincide with the period of cell division that occurs after pollination. At 35 DAA cell division has ceased, the rate of cell expansion increases and starch accumulation begins. 60 DAA coincides with the greatest rate of cell expansion and starch accumulation. By 87 DAA the rate of cell expansion has declined but cell expansion continues at a reduced rate until full ripeness, starch levels peak shortly after this timepoint. In the year in which the samples were taken harvest ripeness was at 132 DAA, at this stage starch levels are rapidly declining and fruit sugars increasing, skin colour is still changing and while some flavour compounds are present full "apple flavour" has not yet developed. By 146 DAA fruit were "tree ripe" at this stage fruit have strong colour and have fully developed flavour, almost all the starch present has been converted into fruit sugars and some flesh softening has occurred. While developmental events that occur prior to full bloom are significant in the developmental program leading to the final fruit, samples prior to full bloom were not considered in this work. RNA was extracted from samples from both replicates, labelled and hybridized to an array of 15726 oligonucleotides (45–55 bases long) designed from 15145 unigenes representing approximately 13000 genes. All samples were compared (using a dye swap design) to genomic DNA (gDNA) as a common reference, making samples directly comparable, the absolute expression of all the samples is shown in Additional file 1.

Figure 1

Apple fruit development

Apple fruit development. Apple fruit at various stages of development. A, 0 DAA, B, 14 DAA, C, 35 DAA, D, 60 DAA, E, 87 DAA, F, 132 DAA, G, 146 DAA. H, diagram of fruit development showing the timing of major physiological events and the sampling time points, adapted from [17–19]. Ripening is shown as a solid and dashed red, solid from the time of the climacteric and dashed for events prior to the climacteric. Bar = 1 cm.

Additional file 1

Array data. An Excel spreadsheet containing expression data for each array feature. Data is shown as mean, number of data points (n) and standard error (SE) of expression levels for the two biological and two technical replicates for all samples except 0 DAA where only one biological replicate was sampled. In some cases one or more data points were excluded from analysis for technical reasons in which case the mean and standard error is calculated from the remaining data. Raw data is lodged with GEO.

Click here for file

Four major groups of co-ordinately expressed genes during fruit development

To examine global changes in gene expression, 8719 genes which changed in expression during fruit development (genes with greater than 5-fold change were excluded in order to see the pattern from genes exhibiting smaller changes, inclusion of these genes did not alter the pattern of expression seen for the majority of genes) were grouped using hierarchical clustering and visualized by plotting expression in 3-dimensional space (Figure 2A and 2B). This global analysis of the microarray shows four major patterns of coordinated gene expression. A group of genes was identified with expression in floral buds but are down-regulated throughout fruit development, a second group of genes was up-regulated early in development and down-regulated later, two additional groups of genes were up-regulated during the middle stages of development and during ripening. By contrast with the results seen for tomato 13, there was no sharp change in global expression patterns at ripening, but this difference is likely to reflect differences in sampling.

Figure 2

Clustering of genes changing during fruit development

Clustering of genes changing during fruit development. Cluster analysis of gene expression. A and B, Expression patterns for the whole array were clustered and then plotted in 3-D space (MATLAB, version 6.0; The Mathworks). Genes with no expression changes or with greater than 5 fold changes were excluded, leaving 8719 genes. y-axis shows fold change. C, The 1955 developmentally regulated genes selected by ANOVA (FDR = 0.01) were clustered by their geometric means. Vertical lines represent transcript level observed for each EST from 0 to 146 DAA, minimum expression (yellow), maximum (red). Major clusters are: floral bud or full bloom (FB); early fruit development (EFD); mid-development (MD); and ripening (R). The EFD and R clusters were further sub-clustered and indicated by EFD1, EFD2, R1, R2 and R3.

To identify those genes that changed expression significantly, a one way ANOVA (model y = time) was applied to the entire dataset. Using a non-adaptive false discovery rate (FDR) control 26 of 0.01, 1986 features were identified (corresponding to 1955 genes) where gene expression changed significantly during fruit development. Hierarchical clustering identified four groups of genes with similar patterns of expression during fruit development (Figure 2C, and Additional file 1, which lists the entire dataset). The full bloom (FB) cluster contained 314 genes (315 features) with high expression at 0 DAA and then low expression during the rest of fruit development. The early fruit development (EFD) cluster contained 814 genes (819 features) where expression peaked between 14 and 35 DAA. The EFD cluster consisted of two weaker sub-clusters: EFD1, a group of 320 genes (326 features) which had high expression early and then very low expression later in development; and EFD2 a group of 493 genes (493 features) with high expression early and moderate expression later in development. The mid development cluster (MD) contained 168 genes (169 features) with expression peaking at 60 and 87 DAA and low expression at other stages of development. The ripening cluster (R) contains 668 genes (681 features) with expression low initially and eventually peaking late in fruit development. The R cluster could be clustered into three further sub-clusters: R1 70 genes (70 features) where expression peaked at harvest ripe (132 DAA) and was low at other stages of development; R2 191 genes (195 features) where expression was very low throughout development until tree ripe (146 DAA); and R3 406 genes (408 features) where expression peaked at tree ripe (146 DAA) but some expression was present at earlier stages of development. Both approaches to clustering identified four major groups of co-ordinately expressed genes suggesting these correspond to major phases of fruit development.

Validation of microarray expression by quantitative RT-PCR

To examine the reliability of gene expression patterns identified from the microarray we used quantitative reverse transcriptase-PCR (qRT-PCR) to examine steady-state RNA levels during fruit development. Genes for qRT-PCR were initially selected from the list of genes that significantly changed their expression during fruit development. The list of regulated genes was ordered from most significant to least significant and genes for qRT-PCR selected at regular intervals from this list (approximately every 50^thgene). Several genes were also chosen for qRT-PCR to confirm expression patterns of genes in particular pathways (see below). Three housekeeping genes were used to normalize qRT-PCR results: an actin gene (Genbank accession CN927806); a GAPDH gene (Genbank accession CN929227) and a gene of unknown function which was selected on the basis of low variability in microarray experiments (Genbank accession CN908822). qRT-PCR expression profiles were compared with microarray expression profiles (Figure 3) and scored as matching if they agreed at all developmental stages or if the majority of stages were in agreement and the significant changes in expression also agreed. By these criteria 74% (26 out of 35) of genes had the same pattern of expression in the microarray experiment as in the qRT-PCR experiment. Interestingly no relationship was observed between the reproducibility of the expression pattern and the significance of the microarray data as determined by ANOVA.

Figure 3

Validation of array expression patterns

Validation of array expression patterns. The pattern of expression for a selection of ESTs was confirmed by quantitative RT-PCR using primers designed close to the array oligo. Graphs show transcript levels from the array (solid lines) for Rep1 (filled diamonds) and Rep2 (open squares) compared with transcript levels from qRT-PCR (dashed lines, mean and standard error for each sample) for Rep1 (filled diamonds) and Rep2 (open squares). X axes show DAA, the left Y axes show relative qRT-PCR expression, the right Y axes show absolute array expression. The genbank accession is shown for each EST.

Genes in different functional classes are expressed at different times during fruit development

To examine the changes in gene function that were occurring during fruit development, functional classes for the apple genes were identified using the Arabidopsis protein function classification defined by the Munich Information center for Protein Sequences (MIPS, using the funcat-1.3 scheme 27). For all the apple genes represented on the array, the Arabidopsis gene with the best sequence similarity based on BLAST analysis was selected 28, with a threshold expect value of 1 × e^-5, and MIPS functional categories for that Arabidopsis gene assigned to the apple gene. This relatively non-stringent threshold was chosen in order to obtain functional classifications for the majority of apple genes on the array. Table 1 shows the number of apple genes, the number of genes with Arabidopsis matches, the number of matches to unique Arabidopsis genes and the number of MIPS functional categories for the entire array, for the 1986 features selected as changing during fruit development, and for the clusters and sub-clusters.

Table 1

Distribution of array features

Subset/cluster^a

ESTs^b

Apple genes^c

Apple genes with hit to Arabidopsis^d

Unique Arabidopsis genes^e

Functional categories^f

whole array

15726

15145

11949

8256

63732

Selected 1986

1983

1955

1442

1330

7523

315

314

225

212

1141

EFD

819

812

603

566

3042

169

168

126

124

653

681

668

495

474

2722

EFD1

326

320

236

220

1128

EFD2

493

368

356

1916

300

195

191

154

885

408

406

284

277

1552

The table shows the number of genes on the whole array and within the clusters as well as the number of Arabidopsis homologues and the number of MIPS function classifications identified.

a FB = full bloom; EFD = Early fruit development; MD = Mid-development; R = ripening; R1, R2, R3 = Ripening subclusters 1, 2 and 3; EFD1, EFD2 = early fruit development subclusters 1 and 2.

b The number of apple ESTs represented by the features on the array.

c The number of apple genes, tentative contigs or singletons identified by the ESTs on the array.

d Apple genes were compared with the Arabidopsis predicted protein set using BLASTx to identify similar Arabidopsis genes, the best match (with expect value better than 1 × e^-5) was used for subsequent functional analysis.

e The number of unique Arabidopsis genes identified by BLASTx using the apple genes, in many cases multiple apple genes had strongest similarity to the same Arabidopsis gene, thus fewer Arabidopsis genes were identified than apple genes.

f Functional categories found for the Arabidopsis genes were identified using the MIPS dataset funcat 1.3.

The distribution of functional categories for the entire array is shown in Table 2 and compared with the distribution of the 1955 genes selected as changing significantly during fruit development, the major clusters and the sub-clusters. The distribution of MIPS functional categories changes between the whole array and the genes selected as changing during fruit development suggest that the genes selected are not a random selection from the array as a whole. For example, there appears to be a higher representation of genes associated with metabolism in the fruit development genes (20.3% vs 16.1% for the whole array) suggesting developing fruit are more active metabolically. Interestingly, there is a slight increase in the unclassified category in the selected fruit development genes 16.7% vs 15.7% for the whole array, while in the ripening cluster the unclassified category is under-represented compared to other clusters (15.2% vs 17.4 to 17.8%), which may reflect the amount of research focused on identifying and characterizing genes involved in the late stages of ripening as compared with early events in fruit development.

Table 2

Functional classification

Mips code^a

Whole array^b

selected

EFD

EFD1

EFD2

Metabolism

16.1

20.3

21.5

20.1

17.2

20.9

18.3

21.1

21.7

25.4

18.4

Energy

2.9

3.4

3.0

2.8

2.1

4.5

2.2

3.1

3.0

5.0

4.4

Cell Cycle and DNA processing

2.9

2.5

1.8

3.4

1.4

1.9

3.3

3.5

0.7

1.9

2.4

Transcription

5.2

4.1

4.3

4.1

3.9

4.4

4.0

3.3

3.1

4.6

Protein synthesis

2.0

1.7

1.5

1.6

1.8

2.0

1.5

1.6

2.7

0.7

2.6

Protein fate

6.6

5.4

4.6

5.0

5.5

6.0

4.5

5.3

4.0

4.7

7.1

Cellular transport & mechanisms

2.4

1.7

1.8

1.6

2.6

1.7

2.1

1.3

0.7

1.7

1.8

Cellular comm/signaling

6.4

5.6

6.5

5.5

5.1

5.6

5.9

5.4

9.0

5.6

4.9

Cell rescue, defense & virulence

3.6

4.0

4.1

4.6

3.6

3.3

4.6

5.7

4.3

2.9

Regulation of/interaction with cellular environment

1.7

1.6

2.1

1.7

2.8

1.1

1.7

0.3

1.1

1.3

Cell fate

3.2

2.6

2.3

2.5

1.5

3.2

2.4

2.6

3.7

2.6

3.3

Systemic regulation of/interaction with environment

1.1

1.3

1.5

1.3

1.1

1.9

0.9

1.3

1.1

Development

1.0

1.2

1.4

1.1

0.9

1.2

1.5

2.0

0.6

0.8

Transposable elements, viral and plasmid proteins

0.1

0.0

0.1

0.0

0.1

0.0

0.2

0.0

Control of cellular organisation

2.7

3.2

2.7

3.8

4.6

2.4

4.8

3.3

2.7

2.3

2.4

Subcellular localisation

19.1

18.1

15.9

17.4

19.8

19.2

17.5

17.4

16.0

18.0

20.5

Protein activity regulation

0.0

0.1

0.2

0.1

0.0

0.2

0.1

0.0

Protein with binding function or cofactor requirement

3.2

2.9

2.7

2.8

4.4

2.8

2.3

3.1

2.3

3.1

2.7

Storage protein

0.1

0.0

0.1

0.0

0.1

0.0

0.1

0.0

Transport facilitation

3.9

3.6

4.4

3.2

2.9

3.7

3.4

3.1

3.0

4.3

3.7

Unclassified

98 or 99

15.7

16.7

17.8

17.4

17.5

15.2

19.1

16.4

18.0

14.2

15.1

The table shows the distribution of classifications as a percentage of the total number of classifications.

a Apple genes for each EST on the array were used to identify Arabidopsis homologues using BLAST with a cutoff of 1 e^-5. Where a putative homologue was identified, the Arabidopsis MIPS (Munich Information centre for Protein Sequences, funcat version 1.3) classification(s) for that gene were applied to the apple EST.

b For the whole array, for the features selected as changing during fruit development, and for each of the clusters and sub-clusters the frequency of occurrence for each functional category is shown as a percentage of the total number of functional categories for that cluster (or sub-cluster). FB = Full bloom; EFD = early fruit development; MD = mid-development; R = ripening; R1, R2, R3 = the 3 ripening sub-clusters; EFD1, EFD2 = the 2 early fruit development sub-clusters.

Within the four major clusters, the genes with peak expression in mid-development have a reduced representation of genes associated with metabolism (17.2% vs 20.1 to 21.5%) suggesting this stage of fruit development might be less metabolically active or use fewer different metabolic genes. In contrast, cellular transport and transport mechanism functions are more highly represented in the mid-development cluster (2.6% vs 1.6 to 1.8%) at the time when fruit are taking up nutrients and water most rapidly.

Control of cellular organization functions are represented more in the EFD and MD clusters (3.8% and 4.6% vs FB2.7% and R2.4%) consistent with this period being a stage of fruit development where the structure of the fruit cells is changing rapidly. In the ripening cluster there is an over-representation of genes in the "energy" category (4.5%) with the lowest representation in mid-development (2.1%). In addition the R2 (peak expression at tree ripe) sub-cluster is over-represented (compared with the other ripening sub-clusters, R1 and R3) in the "metabolism" category (25.4% vs 21.7 and 18.4%) correlating with changes in energy and metabolism during late ripening.

One feature of note was the higher proportion of genes with a cell cycle classification in the EFD cluster (FB 1.8%, EFD 3.4%, MD 1.4%, R 1.9%). The EFD cluster contains genes for which expression peaks in the first 30 days of fruit development, the stage of development when cells are dividing 1718. This developmental period involves the division of specific cells to form the final apple fruit shape and since there appeared to be an increase in cell cycle associated genes during this period we identified the genes associated with the cell cycle classification for each cluster (FB 17 genes, EFD 61 genes, MD 8 genes, R 42 genes) and their annotations (Table 3). These lists are likely to include those genes important in the regulation of fruit size and shape. For example, analysis of these lists identified three core cell cycle genes (see below), which will be the focus of future research.

Table 3

Annotation of cell cycle genes by cluster

FB cluster

EST

Genbank acc.

Best A. thaliana hit^a

e value

Description^b

5019

CN936403

AT5G44680.1

1e-40

methyladenine glycosylase family protein

5126

EB107042

AT2G38620.1

9e-80

CDKB1;2 cell division control protein

33679

CN929052

AT2G47420.1

9e-18

dimethyladenosine transferase

59120

CN862228

AT5G42320.1

2e-12

zinc carboxypeptidase family protein

67405

CN864463

AT5G53000.1

3e-31

protein phosphatase 2A-associated 46 kDa protein

86932

EB119954

AT1G01490.1

2e-19

heavy-metal-associated domain-containing protein

124169

CN937737

AT1G18660.1

3e-67

zinc finger (C3HC4-type RING finger) family protein

134415

CN888558

AT3G62600.1

1e-153

DNAJ heat shock family protein

140667

CN938500

AT2G24490.1

8e-46

replication protein, putative

222173

CN876164

AT4G11010.1

9e-47

nucleoside diphosphate kinase 3, mitochondrial (NDK3)

226032

EG631233

AT3G08500.1

3e-48

myb family transcription factor (MYB83)

254247

CN912925

AT1G10290.1

3e-49

dynamin-like protein 6 (ADL6)

256645

EB151655

AT1G79350.1

1e-77

EMB1135 DNA-binding protein, putative

257305

CN908171

AT3G57550.1

3e-41

guanylate kinase 2 (GK-2)

258270

CN914773

AT2G30110.1

1e-179

ubiquitin activating enzyme 1 (UBA1)

264677

CN910366

AT3G48160.2

6e-68

E2F-like repressor E2L3 (E2L3)

264992

CN917058

AT5G23430.1

1e-53

transducin family protein/WD-40 repeat family protein

EFD cluster

EST

Genbank acc.

Best A. thaliana hit

e value

Description

12163

EB109178

AT3G28030.1

2e-27

UV hypersensitive protein (UVH3)

14094

CN931474

AT2G01440.1

6e-15

ATP-dependent DNA helicase, putative

15274

CN932236

AT3G25500.1

8e-26

FH2 domain-containing protein

19893

CN925129

AT1G73540.1

3e-11

ATNUDT21 MutT/nudix family protein

29516

EB111254

AT2G39730.1

9e-72

RuBisCO activase

31066

CN927871

AT3G23890.1

8e-13

DNA topoisomerase II

33027

CN928590

AT3G25500.1

3e-39

FH2 domain-containing protein

43417

EB113579

AT1G69770.1

3e-06

chromomethylase 3 (CMT3)

45185

CN857495

AT5G05510.1

2e-25

low similarity to SP:O60566 Mitotic checkpoint serine/threonine-protein kinase BUB1 β

62518

EB116342

AT3G08910.1

7e-67

DNAJ heat shock protein

64262

CN850169

AT2G30200.1

1e-148

T27E13_6

85474

CN869267

AT1G68760.1

6e-54

ATNUDT1 MutT/nudix family protein

91885

CN871666

AT1G10520.1

3e-15

DNA polymerase lambda (POLL)

93419

CN874495

AT5G26751.1

4e-58

shaggy-related protein kinase α/ASK-α (ASK1)

95093

CN875141

AT5G18110.1

5e-60

novel cap-binding protein (nCBP)

105540

CN886787

AT3G51770.1

1e-111

similar to tetratricopeptide repeat (TPR)-containing protein

111728

EB124553

AT1G44900.1

3e-50

DNA replication licensing factor

118006

EB125634

AT2G21790.1

8e-45

ribonucleoside-diphosphate reductase small chain, putative

119405

CN887179

AT1G68010.1

1e-81

glycerate dehydrogenase/NADH-dependent hydroxypyruvate reductase

120390

CN890521

AT1G21660.1

7e-12

low similarity to SP:O14976 Cyclin G-associated kinase

138266

CN937814

AT2G17120.1

3e-79

peptidoglycan-binding LysM domain-containing protein

142020

CN939277

AT2G38810.1

2e-48

histone H2A, putative

142920

EB127800

AT5G57850.1

2e-08

aminotransferase class IV family protein

148629

EB138792

AT3G22630.1

2e-36

20S proteasome β subunit D (PBD1) (PRGB)

149453

CN897394

AT5G55230.1

1e-118

ATMAP65-1 Binds and bundles microtubules

149668

CN897544

AT4G36080.1

1e-103

FAT domain-containing protein/phosphatidylinositol 3- and 4-kinase family protein

151134

EB139596

AT2G42580.1

5e-24

tetratricopeptide repeat (TPR)-containing protein

151602

CN898773

AT5G13780.1

8e-81

GCN5-related N-acetyltransferase, putative, similar to ARD1 subunit

152213

CN940414

AT2G35040.1

1e-112

AICARFT/IMPCHase bienzyme family protein

153604

EB140203

AT1G55350.1

EMB1275 calpain-type cysteine protease family

153992

CN900578

AT2G21790.1

1e-160

R1 ribonucleoside-diphosphate reductase small chain, putative

155385

CN901052

AT2G21790.1

2e-83

R1 ribonucleoside-diphosphate reductase small chain, putative

155966

CN901211

AT5G61060.1

2e-34

histone deacetylase family protein

159200

CN940759

AT2G14880.1

6e-36

SWIB complex BAF60b domain-containing protein

162529

CN942994

AT3G44110.1

1e-152

DNAJ heat shock protein, putative (J3)

163128

CN943384

AT1G20930.1

1e-102

CDKB2;2 cell division control protein, putative

163154

CN943405

AT5G61060.1

2e-84

histone deacetylase family protein

166835

EE663942

AT3G17880.1

1e-58

tetratricoredoxin (TDX)

170408

EB140959

AT3G08910.1

7e-59

DNAJ heat shock protein, putative

170963

CN882668

AT2G46225.1

2e-20

ABI1L1 Encodes a subunit of the WAVE complex

171493

CN883039

AT2G29570.1

1e-111

PCNA2 proliferating cell nuclear antigen 2 (PCNA2)

172325

CN883596

AT5G08020.1

7e-91

similar to replication protein A1 (Oryza sativa)

173799

EB141951

AT2G27960.1

6e-37

CKS1 cyclin-dependent kinase

180731

CN904791

AT1G75690.1

2e-55

chaperone protein dnaJ-related

181072

CN904980

AT3G18190.1

chaperonin, putative

184975

EB148197

AT5G44680.1

1e-90

methyladenine glycosylase family protein

186444

EB149644

AT3G19420.1

2e-12

MLD14.22

186960

EB150084

AT3G08690.1

9e-27

ubiquitin-conjugating enzyme 11 (UBC11), E2

213416

EB157314

AT1G62990.1

1e-126

homeodomain transcription factor (KNAT7)

220588

EB132350

AT3G48590.1

2e-15

CCAAT-box binding transcription factor Hap5a, putative

220604

CN948726

AT4G33260.1

8e-17

WD-40 repeat family protein

245977

CN903005

AT3G26730.1

1e-49

zinc finger (C3HC4-type RING finger) family protein

256235

CN913864

AT2G31320.1

NAD(+) ADP-ribosyltransferase, putative

256449

CN916743

AT3G22890.1

1e-165

sulfate adenylyltransferase 1/ATP-sulfurylase 1 (APS1)

257853

CN914478

AT5G52640.1

heat shock protein 81-1 (HSP81-1)

261756

CN908391

AT2G25050.1

5e-07

formin homology 2 domain-containing protein

264654

CN910347

AT5G67100.1

5e-87

DNA-directed DNA polymerase α catalytic subunit, putative

265667

CN910570

AT5G16270.1

3e-06

Rad21/Rec8-like family protein

266414

EB152178