Role of the KNOTTED1-LIKE HOMEOBOX Protein (KD1) in regulating tomato flower pedicel abscission at early and late stages of the process

Background The KNOTTED1-LIKE HOMEOBOX PROTEIN1 ( KD1 ) gene is highly expressed in flower and leaf abscission zones (AZs). RNA-antisense silencing of KD1 was shown to delay tomato pedicel and petiole abscission, induced by flower or leaf removal, respectively. KD1 was found to regulate flower pedicel abscission via alteration of auxin gradient through the flower AZ (FAZ), and disruption of the auxin response at the early stages of the abscission process. The present work was aimed to further understand how KD1 regulates signaling factors and regulatory genes involved in the delay of pedicel abscission using the silenced KD1 lines. For this purpose we performed a large scale transcriptome profiling of the FAZ at various time points after flower removal, using a customized AZ-specific microarray. Results The results highlighted a differential expression of regulatory genes in the FAZ of KD1 silenced plants compared to the wild type (WT). These genes were controlled by KD1 before and after abscission induction. In the KD1 -silenced plants, KD1 expression already decreased at zero time before flower removal, resulting in altered expression of regulatory genes, including epigenetic modifiers, transcription factors (TFs), post-translation regulators, and antioxidative defense components. The increased expression of these regulatory genes, and genes related to exocytosis and gibberellin perception, observed in the WT FAZ, was inhibited in the KD1- silenced plants at 4 h after flower removal. This response led to an inhibited abscission phenotype and downregulation of genes involved in abscission execution and defense in the KD1 -silenced plants. Conclusions The data suggest that KD1 is a master regulator in tomato flower abscission. Some of the altered genes might directly affect auxin homeostasis and transport, resulting in slowing down auxin depletion in the FAZ. This probably delayed the subsequent cascade of the molecular events described above. The data obtained suggest that the inhibitory effect of KD1 silencing on flower pedicel abscission is not limited to manipulation of auxin levels and response as previously reported, but it probably also the Ca 2+ /CaM signal transduction.

separation and proximal-distal axis development in leaves of dicots [12][13][14]. In monocots, the KNOX regulatory network was reported to play a role in shoot apical meristem maintenance in rice and maize [15,16]. In different plant systems, the class I KNOX family proteins were found to regulate several genes involved in biosynthesis and signal transduction of various plant hormones, various TFs and lignin synthesis [17]. Recently, a KNOXI transcription factor (TF) was demonstrated to regulate floral fate in Arabidopsis, further clarifying the role of KNOX TFs in the specification of flower meristem identity [18].
Manipulating KD1 expression affected various aspects of plant development, which were all related to the KD1 role in auxin gradient responses [19], and the KNOX protein Knotted1 was reported to directly control the auxin signaling pathway in maize at all levels, including auxin synthesis, transport, and signaling [20]. Indeed, our previous work, which investigated the role of KD1 in abscission, demonstrated a strong effect of KD1 on the auxin gradient in the FAZ [6]. Thus, the KD1 gene was silenced, under the control of the AZ-specific promoter, Tomato Abscission Polygalacturonase4 (TAPG4) [2,6,21,22], to study the changes in gene expression profiles in the tomato FAZ shortly (4 h) after flower removal, in the TAPG4::antisense KD1 plants compared to the wild type (WT). The data obtained showed that the involvement of KD1 in abscission regulation was associated with auxin transporters and signaling components, and that the changes in KD1 expression modulated the auxin concentration and response gradient in the FAZ. Reducing KD1 transcript abundance, which is highly expressed in the AZs, delayed organ abscission, but it did not prevent the process, suggesting that KD1 is part of a complex system controlling the abscission process [6].
Our previous study [6] raised some questions that remained unsolved. These included: a) how KD1 is regulating the auxin-related genes; b) what is the role of KD1 in regulation of other signaling factors and regulatory genes involved in the delay of pedicel abscission at zero time and at the early (4 h) and late (8-20 h) stages of the abscission process occurring after flower removal. To answer these questions, we performed in the present study a detailed transcriptome analysis of the FAZ and the NAZ of WT plants compared to the FAZ of the TAPG4::antisense KD1 plants, at various time points after flower removal, using a newly developed customized AZ-specific microarray [22]. The use of this customized AZ-specific microarray in a time course analysis, allowed to expand the database of changes in gene expression occurring at the early stage of pedicel abscission, since it contains more gene probes than those that were previously used by Ma et al. [6]. The results obtained in the present study highlighted expression of regulatory genes in the FAZ, which seem to be controlled by KD1 before and after abscission induction. These genes might have a role in regulating the acquisition of the FAZ cell competence to respond to ethylene signaling, or in controlling abscission in a different mode of action.
Results And Discussion Downregulation of KD1 in the AZ inhibits tomato pedicel and petiole abscission Quantitative reverse transcription (qRT-PCR) demonstrated that the KD1 transcript was predominately expressed in the FAZ, and its expression was downregulated to very low levels within 8 h after flower removal ( Fig. 1). This rapid decrease in KD1 expression in the tomato FAZ after flower removal was already demonstrated in our previous publications [2,6]. Pretreatment with the ethylene inhibitor 1methycylopropene (1-MCP) did not affect the expression of KD1, but application of IAA after flower removal prevented the decrease in its expression [2]. These data indicate that KD1 expression in the FAZ is IAA-dependent, and that the decrease in its expression is a result of IAA depletion after flower removal. Similar results were reported for other Knotted family members, such as Knotted TKN2/LET6 (AF000141) and knotted TKN4 (AF533597) [2]. The involvement of three knotted proteins, KNAT1, KNAT2, and KNAT6, was also reported for the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-HAE/HSL1-dependent flower organ abscission in Arabidopsis [8][9][10]. These observations suggest that KD1 and TKN4 could function as regulators of the acquisition of AZ cell competence to respond to ethylene signaling after IAA depletion.
The important role of KD1 in tomato flower and leaf abscission, was previously demonstrated by virusinduced gene silencing (VIGS), andby antisense silencing under the AZ-specific promoter TAPG4, which led to delayed pedicel and petiole abscission [6]. The regulation of abscission by KD1 was found to be associated with modulation of the auxin concentration and response in the FAZ, leading to changes in the abundance of genes related to auxin transporters and signaling components [6]. We used the same lines A and E of generation T4 in the present study.
The efficacy of KD1 silencing is demonstrated by the qRT-PCR results ( Fig. 2A). It is evident that the TAPG4 promoter was very active in the FAZ [21], and KD1 expression was downregulated by about 60% at zero time, and by about 70% at 4 h after flower removal, approximately at the same rates that were previously reported [6]. The microarray antisense probe showed increased expression of KD1 between 4-20 h after flower removal (Fig. 2B). This antisense probe spanned the cloned fragment of KD1 (Fig. 2C). We expected a higher expression of the antisense probe also at zero time The inhibition of petiole abscission in the transgenic plants was more significant in leaf position 4 ( Fig.   3C), and also in leaf positions 2 and 3 (data not shown). The inhibition of petiole abscission after leaf deblading and ethylene treatment indicates that the petiole AZ cells in the KD1-silenced plants were less sensitive to ethylene treatment after auxin depletion. This supports our hypothesis that KD1 participates in regulating the AZ cell competence to respond to ethylene signals.
The molecular events in the process of tomato pedicel abscission were divided into two phases: early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) [2]. It is important to note that the previous transcriptomic experiment was performed with Solanum lycopersicum, cv. 'Shiran', in which 100% of pedicel abscission was obtained already at 14 h after flower removal, and therefore, the time point of 8 h was considered as a late stage. In the present study, we used Solanum lycopersicum, cv. 'New Yorker', in which 100% of pedicel abscission was obtained after 20 h (Fig. 3A), and therefore the time point of 8 h is considered as an early stage in the  Table 1). The complete list of differentially expressed genes was generated and presented in Additional files 1-6: Tables S1-S6. Our microarray results reconfirmed the gene expression profiles reported by Ma et al. [6] in the FAZ at 4 h after flower removal, and revealed the potential role of other regulatory genes associated with KD1 at the early stage of the pedicel abscission process (Figs. [4][5][6][7][8]. To validate the microarray assay results, we selected the following five genes, Cellulase1 (Cel1) Therefore, we focused mainly on genes, which showed a modified expression in the FAZ before flower removal (zero time) and at 4 h after flower removal. This early period represents the timing in which the FAZ cells acquire the competence to respond to ethylene signals, which initiates the abscission process [1,2,7]. The next abscission phase, the execution stage, which starts during the period of 4 to 8 h after flower removal, can also be regulated both by signals derived from the previous stage, as well as by signals generated during this stage, that cannot be distinguished from one another.
The results presented in Our results revealed that TAPG::antisense KD1 silencing altered the expression of the following major regulatory genes specifically in the FAZ at different levels of regulation: four genes related to plant epigenetics; two MADS-box homeobox TFs; eight post translation regulation genes related to ubiquitin-based protein degradation or phosphorylation/de-phosphorylation; and four ethylene-related genes. Some of these altered genes might trigger a cascade of molecular events, leading to alteration of the auxin levels and response in the FAZ at the early stages of the abscission process, resulting in a reduced competence of the FAZ cells to respond to ethylene, as demonstrated in a previous study [6]. Similarly, a reduced competence of the leaf AZ to respond to ethylene after leaf deblading, was also obtained (Figs. 3B, 3C). The four ethylene-related genes, three ACO genes ( The expression of the TF gene encoding the Plant Homeodomain (PHD)-finger family protein, which was specifically upregulated in the WT FAZ at 4 h after flower removal and remained high up to 16 h, was significantly inhibited in the FAZ of the silenced plants (Fig. 6C). The PHD finger protein has a metal binding RING domain (Cys3-His-Cys4) motif. The PHD domain has a conserved Zinc finger (Znf) domain in eukaryotic organisms. PHD finger in proteins related to epigenetics are involved in the interaction between proteins, especially the modification on histone of nucleosome, such as methylation, acetylation, and phosphorylation [24]. A similar expression pattern, i.e. upregulation in the WT FAZ and inhibition in the FAZ of the silenced plants, was also observed for two bHLH and Znf TF genes, SlbHLH048 and SlbHLH046 (Fig. 6D1, D2). Some bHLH and Znf TF genes were previously reported to be involved in abscission of olive fruit and tomato flower pedicels [2,25].
Six genes related to the Ca 2+ signal transduction, two kinases and four Ca 2+ /Calmodulin (CaM)related, which were upregulated at 4 h after flower removal in the WT FAZ, were inhibited in the FAZ of the silenced plants ( Fig. 6E1-E4, F1-F2). Calcium ions (Ca 2+ ) serve as a universal messenger involved in the modulation of diverse developmental and adaptive processes in response to various physiological stimuli [26,27]. Our results showed that the Ca 2+ /CaM-mediated signal transduction plays a role in regulating the abscission process, and it is probably regulated by KD1. Previous reports on various abscission systems demonstrated the involvement and importance of Ca 2+ /CaM signaling in regulation of organ abscission. Thus, a cycle of water stress/rehydration, which induced citrus leaf abscission, resulted in upregulation of a CaM gene in the laminar leaf AZ at 1 h after rehydration [28].
Induction of litchi fruitlet abscission by ethephon treatment resulted in expression changes of genes related to calcium transport and perception. Hence, 19 and 33 transcripts were up-and downregulated, respectively, following the ethephon treatment, including CNGC genes [29]. Similarly, maturation of olive fruits, which induced their abscission, caused upregulation of CaM, CML, and Calcium-binding protein kinase genes in the fruit AZ [25]. Our results demonstrate that in the system of tomato pedicel abscission induced by flower removal KD1 silencing inhibited the upregulation of Ca 2+ /CaM signaling-related genes. These findings support the previous reports regarding the involvement of Ca 2+ /CaM signaling in organ abscission, and suggest that KD1 plays a significant role in regulating the Ca 2+ /CaM signal transduction.
Of particular interest is the exocytosis-related gene, Syntaxin, which encodes a membrane integrated protein, Q-Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE), necessary for vesicle trafficking. This gene, which was upregulated in the WT FAZ, was significantly downregulated in the FAZ ofthe silenced plants (Fig. 6F3). The primary role of SNARE proteins is to mediate vesicle fusion with their target membrane bound compartments participating in exocytosis [30]. Previous reports showed that mobilization of the secretory pathway leads to the release of cell wall modifying enzymes to implement abscission [31,32]. Additionally, Agusti et al. [28] reported the induction of several genes involved in vesicle trafficking, such as SNARE-like protein and Syntaxin, in citrus laminar AZ during leaf abscission induced by a cycle of water stress/rehydration. Analysis of gene expression in the melon fruit AZ revealed that a sequential induction of cell wall-degrading genes was associated with the upregulation of genes involved in endo-and exocytosis during mature fruit abscission [33].
Our analysis showed that the ABC transporter gene, an ATP-binding cassette transporter which belongs to the ABCA sub-family, was downregulated in the KD1-silenced plants compared to the WT (Fig. 6F4). This gene was shown to be involved in auxin transport, and is specifically expressed in the root system [34]. Similarly, the Coatomer beta subunit gene was also downregulated in the silenced plants (Fig. 6F1). The coat protein complex genes are responsible for reverse transport of recycled proteins from the Golgi and pre-Golgi compartments back to the ER and vice versa [35].
In the present work, two Cytochrome P450 genes were downregulated in the FAZ of the KD1-silenced plants up to 16 h after flower removal, reaching a similar level of expression to that observed in the WT FAZ at 20 h (Fig. 6G1, G2). Another Cytochrome P450 genewas also downregulated in the silenced plants up to 12 h after flower removal, and reached a similar level of expression as in the WT FAZ during 12-20 h (Fig. 8D). In Arabidopsis, the cytochrome P450s were shown to be involved in catalyzing the first step of tryptophan-dependent IAA biosynthesis [36]. The involvement of auxin and auxin-related gene expression in pedicel abscission of the WT and the KD1-silenced plants was reported previously [6].
Gene expression of several oxidase/reductase-related genes, such as Laccase (Fig. 6G3), Ascorbate Oxidase (AO) Fig. 6G5), Nitric Oxide (NO) reductase (Fig. 6G6), and Alcohol Dehydrogenase (Fig. 6H) was downregulated in the FAZ of the silenced plants, whereas in the WT FAZ these genes were upregulated during the abscission process. This suggests the involvement of oxidative processes in pedicel abscission.
C3. Genes whose expression was specifically-and transiently-altered in the FAZ of KD1-silenced plants at 8 h after flower removal Several genes were specifically and transiently altered in the FAZ of KD1-silenced plants at 8 h after flower removal, when the execution of cell separation had already started (22% of pedicel abscission was obtained in the WT- Fig. 3A). One set of genes represents diverse gene families that were transiently upregulated at 8 h in the silenced plants compared to the WT (Fig. 7E-L7). These genes included: A) CONSTANS interacting protein6 (Fig. 7E), which was shown to control flowering in response to photoperiod in Arabidopsis [37,38], and leaf induction by cytokinins in tomato [39]. B) RNA-binding La domain protein (Fig. 7F), in which La acts as an RNA polymerase III (RNAP III) TF. C) cDNA clone (Fig. 7G), that also encodes an RNA-binding protein. D) Dopamine-Monooxygenase Nterminal (DOMON) domain-containing protein, also called DoH (Fig. 7J), was originally identified in several secreted or cell surface proteins from plants and animals [40]. It is usually associated with other redox domains in larger proteins such as cytochromes b561, and has a suggested capability of transmembrane electron transport [40][41][42].E) Seven Unknown Protein genes that have a similar expression pattern (Fig. 8L1-L7). Since these genes were upregulated in the FAZ of the silenced plants, in which pedicel abscission was significantly inhibited, unlike the WT in which pedicel abscission had already started (Fig. 3A), some of them might have an inhibitory role in the abscission process. Interestingly, seven stress defense-associated genes had the same expression pattern (Fig.   7K1-K7). However, it is not yet clear why these genes were upregulated in the silenced plants, which showed an inhibited abscission phenotype.

CaM-Like protein
The GFP fused to the DES1 promoter was reported to be highly expressed in the AZ of seeds and siliques, and the des1 mutants exhibited an altered leaf senescence phenotype [50]. H) The gibberellin (GA) receptor gene, GID1L2, which was upregulated in the WT FAZ at 4 h and peaked at 8 h after flower removal (Fig. 8I), suggesting that the inhibition of abscission by KD1 silencing might be mediated also through the inhibition of GIDIL2. I) Some other Unknown Protein genes (Fig. 8L1-L4).

D. KD1 silencing decreases the expression of cell wall modifying genes
It is well established that cell wall loosening and cell separation occurring in the AZ are controlled by distinct sets of cell wall degrading enzymes. Dissolution of the middle lamella or the shared cell wall in the AZ is a fundamental step in the abscission process. Enzymes and proteins associated with disassembly and modification of the cell wall include PGs, cellulases, endoglucanases, pectin methylesterases, pectate lyases, xyloglucan endotransglucosylase/ hydrolases (XTH), and expansins (EXP) [10,[51][52][53][54]. Therefore, the expression pattern of these cell wall modifying enzymes occurring at the late stages of abscission, was used as an additional marker for confirming the effects of KD1 silencing in delaying pedicel abscission.
Our transcriptome analysis revealed a specific upregulation of 28 genes encoding cell wall modifying enzymes in the WT FAZ, whereas in the NAZ the expression of most of them did not change and remained very low during the entire experimental period (Fig. 9A-J). These genes, which belong to 11 families, were downregulated by KD1 silencing, as manifested by their lower or delayed expression in the FAZ of the silenced plants (Fig. 9). Only Cel8, XTH26, and EXP2 genes exhibited some increased expression in the NAZ (Fig. 9B3, C7 and F1). Among the cell wall hydrolyzing enzymes, TAPG4,5, and XTH3a,3c,3d,7,10 were upregulated in the WT FAZ at 4 h after flower removal, and their expression remained high up to 16 h (Fig. 9A3, A4, C1-C5). On the other hand, Glucanase, Pectin Esterase, and EXP genes were upregulated only at 8-12 h after flower removal (Fig. 9E1-J). The tomato AZ-specific TAPG genes, TAPG1,2,4,5, were downregulated in the silenced plants compared to the WT (Fig. 9A1-A4). The expression patterns of TAPG1,2,4 in the WT FAZ were identical to their patterns reported previously [2], thereby confirming the microarray results obtained by the tomato AZ-specific microarray chip. Our results further demonstrate that the organ abscission execution, manifested by upregulation of cell wall degrading proteins at phase C of the abscission process [10,[53][54][55], is a programmed event, in which these proteins were sequentially increased (Fig. 9).

D1. KD1
Phase D of the abscission process, in which the production of a protective defense layer occurs [10,[53][54][55], was shown to significantly overlap with the execution phase C of abscission [23]. We present here only selected data of the numerous defense genes that were specifically upregulated in the tomato WT FAZ, and were significantly inhibited in the KD1-silenced plants. Of the set of genes that were specifically and transiently altered in the WT FAZ at 8 h after flower removal, three genes should be mentioned: Omega-6 Fatty Acid Desaturase (Fig. 8J1) and two Lipoxygenase (LOX) genes, Cevi34/LOX (Fig. 8J2) and LOX (Fig. 8J3). Some of the defense-related genes were upregulated in the WT FAZ very early after flower removal (Fig. 10A-E2; marked with red*), while others were upregulated only later on (Fig. 10E3, E4, F1, F2; marked with green*). The expression of all these genes was inhibited in the KD1-silenced plants (Fig. 10). These genes are related to abscission, as they were upregulated only in the WT FAZ, in which abscission took place after flower removal. These results confirm the overlapping between phases C and D of the abscission process.

Conclusions
Based on the results presented in this study, we could further elucidate how KD1 delays pedicel abscission. The present data indicate that KD1 is a master regulator of tomato pedicel abscission, as the expression of important regulatory genes was modified in the FAZ of the KD1-silenced plants at 4 h after flower removal, i.e. at the early stage of the abscission process, compared to the WT FAZ. This led to the inhibition of pedicel abscission, as summarized in Figure 11. Moreover, it should be noted that KD1 expression decreased in the silenced plants already at zero time, which led to the modified expression of regulatory genes before flower removal (Fig. 11A). These early modified regulatory genes included epigenetic modifiers, TFs, post translation regulators, and antioxidative defense factors. Some of them might directly affect auxin homeostasis and transport, resulting in slowing down auxin depletion in the FAZ, which leads to inhibition of the acquisition of the competence of the FAZ to respond to the ethylene signaling. The changes in gene expression occurring at 4 h after flower removal (Fig. 11B) might result either from a direct effect of KD1 silencing, or from the changes in gene expression occurring at zero time, prior to abscission induction (Fig. 11A). The above data suggest a possible mode of action for the manipulation of auxin levels and response obtained following KD1 silencing, which finally leads to inhibition of pedicel abscission.
The effects of KD1 silencing, which result in the inhibited abscission phenotype, include also indirect effects leading to downregulation of genes involved in abscission execution and defense. Thus, the inhibited expression of exocytosis and protein transporters such as AAA-ATPase and Syntaxin, which inhibited the export of Golgi-derived vesicles containing cell wall degrading enzymes across the plasma membrane of AZ cells, might have an important role in the inhibited abscission phenotype of the KD1-silenced plants. It seems therefore, that the inhibitory effect of KD1 silencing on flower pedicel abscission is not limited to manipulation of auxin levels and response as previously reported, but it probably also operates via alteration of other regulatory pathways that delay the acquisition of the competence of the FAZ cells to respond to ethylene signaling and abscission execution (Fig. 11B).
Since KD1 belongs to the class I KNOX family, its role in organ abscission is in agreement with the various regulatory roles of class I KNOX family proteins, which were found to regulate several genes involved in biosynthesis and signal transduction of various plant hormones, TFs, and lignin synthesis.
Taken together, the present study shades light on the role of KD1 as a master regulator of various signaling factors and regulatory genes involved in organ abscission processes, in addition to its welldocumented involvement in leaf development, shoot apical meristem maintenance, and differentiation of flower meristems.

Plant material and abscission induction treatments
The experiments were performed with tomatoSolanum lycopersicum, cv. 'New Yorker') plants, grown from seeds obtained from the Tomato Genetics Resource Center, University of California, Davis, USA.
All plants were grown in a greenhouse located in The Volcani Center, Israel, under a controlled temperature of 25°C and natural daylight. The flower abscission experiments were carried out with plants grown in 10-L pots. The inflorescences of both WT and the KD1-antisense transgenic lines were harvested from 4-month-old plants as previously described [2,7]. The experiment protocols and pedicel abscission assays were performed in a controlled observation room maintained at 20°C and 60 to 70% relative humidity with continuous light of 14 μmol m -2 s -1 , as previously described [2,7,23]. The number of abscised pedicels was monitored daily by touching the distal side of the FAZ, for calculating the accumulated percentage of pedicel abscission.
Leaf abscission experiments were carried out with plants grown in 576 cm 3 pots, which had 6 to 8 true leaves per plant. For abscission induction, the leaves of the WT and the KD1-silencedtransgenic plants were debladed for auxin depletion, and 48 h after leaf deblading, the plants were treated with 10 µL L -1 ethylene for 24 h at 23°C, as previously described [22].

Vector construction and plant transformation
The generation of the TAPG4::antisense KD1 transgenic plants was basically performed as previously described [6]. Briefly, a 2379-bp fragment of the AZ-specific TAPG4 promoter from tomato genomic DNA and a 227-bp fragment of the KD1 gene from tomato cDNA were amplified, and then sub-cloned into the modified binary vector GSA1285 in an antisense orientation. We used the primers 5'-TCTCAGCTCAGTGAACTCATGG-3' and 5'-TTGTGGCAATCTAGCCA-TACAT-3' for the KD1 amplification, introduced the plasmid constructs into Agrobacterium tumefaciens (LBA4404), and transformed the tomato 'New Yorker' as previously described [56]. In this way we generated 10 independent transgenic lines, from which we selected two representative transgenic lines (lines A and E, T4 generation) as previously reported [6], for further analysis.

Gene expression profiling
For expression and microarray studies, we used samples of the FAZ and the NAZ of the WT and the FAZ of TAPG4::antisence KD1 line E, collected in duplicates at various time points (0, 4,8,12,16, and 20 h after flower removal). The RNA was extracted, treated with DNase, processed and checked for its quality, using the Agilent platform (Agilent, Palo Alto, CA), as previously described [7,22].

Microarray
The experimental procedures for microarray labeling, hybridization, and scanning were performed as previously detailed [7], using the AZ-specific microarray chip, AMADID: 043310 (Genotypic Technology, Pvt. Ltd, Bangalore, India) for hybridization of the labeled cRNA samples [22]. We used the Feature Extraction software of Agilent V-11.5 for the data extraction from images. The analysis of the microarray data, using duplicate samples for each time point, to identify significant genes that were up-and down-regulated within the group of samples, was basically performed as previously detailed [7].

Validation of gene expression by qRT-PCR
All the procedures for validation of gene expression were performed as previously detailed [7], according to the primers sequences, amplicon length, and annealing (Tm) presented in Additional file 8: Table S7. These primers matched the microarray probes, and the qRT-PCR and microarray analyses were performed with the same RNA samples, as previously described [22]. ACTIN was used as the reference gene for determination of the relative expression levels of the identified genes.

Sequence deposition
The data for the TAPG::antisense KD1 FAZ samples (12 arrays) were submitted under the Gene Expression Omnibus database NCBI-GEO accession id: GSE64564. The microarray data for WT (cv. 'New Yorker') FAZ and NAZ samples (12 arrays each) were submitted as previously detailed [7] under accession id: GSE64221. The data will be released for public access upon acceptance of the manuscript.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its Additional files. Any other informationis available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.         Effect of antisense silencing of KD1 on the kinetics in array-measured expression levels of genes encoding cell wall modifying enzymes that were specifically upregulated in the WT FAZ at specific time points after flower removal: early -4 (*) or 8 (*) h or late 12-20 (*) h.