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 1-methycylopropene (1-MCP) did not affect the expression of KD1, but application of IAA after flower removal prevented the decrease in its expression . 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) . 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 [8–10]. 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 virus-induced gene silencing (VIGS), andby antisense silencing under the AZ-specific promoter TAPG4, which led to delayed pedicel and petiole abscission . 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 . 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 , 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 . 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 (Fig. 2B), because the expression of KD1 in the transgenic plants decreased by 60% at this time point (Fig. 2A). At present we do not have a reasonable explanation for this unexpected results.
The antisense transgenic lines exhibited a very significant delay in pedicel and petiole abscission following abscission induction (Fig. 3). All the pedicels in the WT plants abscised at 20 h after flower removal, compared to only 40% of abscised pedicels in the TAPG4::antisense KD1 lines (Fig. 3A). Even at 48 h after flower removal, 25% of the pedicels in the transgenic plants still remained attached (data not shown). Petiole abscission of leaves at position 1 above the cotyledons reached 100% in the WT plants 4 days after leaf deblading and ethylene treatment, whereas in the TAPG4::antisense KD1 plants it reached only 60 and 70% abscission rate in the silenced lines A and E, respectively (Fig. 3B). 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) . 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 pedicel abscission execution process. The early events (0–4 h) probably lead to acquisition of the competence of FAZ cells to respond to ethylene signaling, and to increased endogenous ethylene biosynthesis. The late events include the execution of pedicel abscission and the development of the defense layer .
C. Mode of action of the inhibition of abscission in KD1-silenced plants
The determination of the role of KD1 in the regulation of abscission in the tomato flower model system, based on the microarray results, should be related to changes in the expression of regulatory genes occurring specifically in the FAZ of the silenced plants at the early stages of pedicel abscission. 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 Figures 5–9 are organized according to the pattern of gene expression rather than according to the function of the related proteins. Figures 5 and 6 present genes that were downregulated or upregulated, respectively, at zero time; Figures 7 and 8A present genes that were upregulated or downregulated, respectively, at 4 h after flower removal and later on; and Figures 8B and 9 present genes that were transiently upregulated or downregulated, respectively, mainly at 8 h after flower removal. Figures 10 and 11 present genes encoding cell wall degrading enzymes, as well as defense and boundary layer-related proteins, which function in the late abscission execution phases, C and D, and not in the early regulatory events [10, 23].
C1. Genes whose expression was specifically altered in the FAZ of KD1-silenced plants at zero time
As a result of the decrease in KD1 expression in the FAZ of the transgenic plants at zero time (Fig. 1), FAZ-specific changes in the expression of some regulatory genes already occurred at this time (Figs. 4 and 5). Eight genes were downregulated in the transgenic plants at zero time, and their expression remained low up to the end of the experiment (Fig. 4), and 20 genes were upregulated and remained high or gradually decreased later on (Fig. 5). The downregulated genes included: two MADS-box genes (Fig. 4A1, A2); a High Mobility GroupHMG) TF (HMG-type nucleosome assembly factor) (Fig. 4B); an orthologue gene of the Arabidopsis Receptor-Like Kinase (RLK) (Fig. 4C); Enoyl-CoA Hydratase (Fig. 4D); NAD-specific Glutamate Dehydrogenase (GLDH) (Fig. 4E); and two Unknown Protein genes (Fig. 4F1, F2). All the downregulated genes in the FAZ of the silenced plants were not FAZ-specific, and were also significantly expressed in the WT NAZ (Fig. 4), except for RLK that was expressed at a low level in the WT NAZ (Fig. 4C).
Twenty genes were specifically upregulated in the FAZ of KD1-silenced lines at zero time. These included: Four F-box family protein genes (Fig. 5A1-A3). The Regulatory Particle (RP) RPN3 (Fig. 5A5). Genomic DNA Chromosome 5P1 clone MBG8 (Fig. 5B1). Chromodomain Helicase DNA-binding protein4 (CHD4) (Fig. 5B2). Histone-lysine N-methyl transferase SETDB1 (Fig. 5B3). Three genes related to ethylene biosynthesis—two 1-Aminocyclopropane–1-Carboxylate Oxidase (ACO)-like proteins (Fig. 5C1, C2), and one ACO-homologue (Fig. 5C3); one AP2-like Ethylene Responsive Factor (ERF) (Fig. 5C4). Six Unknown Protein genes (Fig. 5D1-D6), a Serine-Threonine-Protein Phosphatase7 (Fig. 5E), and Glutaredoxin (Fig. 5F). All these genes were significantly upregulated in the FAZ of the silenced lines from zero time up to at least 8 h after flower removal (Fig. 5).
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 . 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 (Fig. 5C1-C3) and one ERF geneFig. 5C4), were unexpectedly upregulated in the FAZ of the silenced plants from zero time up to 20 h. On the other hand, a transiently upregulation of one ACO and two ERF genes in the WT FAZ was inhibited in the FAZ of the silenced plants (Fig. 6B1-B3). The expression pattern in the WT FAZ and NAZ at different time points of other ethylene-related genes involved in its biosynthesis (ACSs and ACOs) and perception (ETRs and CTRs) (data not shown), resembled the previously reported expression pattern for these genes . The expression of these ethylene-related genes as well as the expression of other ERF genes, in the FAZ (21 out of 24 ERFs) after flower removal, was not affected by KD1 silencing (data not shown). These data suggest that the inhibition of pedicel abscission induced by KD1 silencing may not necessarily be ethylene-mediated.
C2. Genes whose expression was specifically altered in the FAZ of KD1-silenced plants at 4 h after flower removal
The differentially regulated genes in the FAZ of the silenced plants at 4 h included various regulatory genes at different levels of regulation: epigenetics; TFs; post-translation such as kinase/phosphatase and protein degradation/ubiquitination; transporters; signal transduction; and oxidase/reductase gene families.
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 . 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 Ca2+ signal transduction, two kinases and four Ca2+/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 (Ca2+) 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 Ca2+/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 Ca2+/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 . 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 down-regulated, respectively, following the ethephon treatment, including CNGC genes . Similarly, maturation of olive fruits, which induced their abscission, caused upregulation of CaM, CML, and Calcium-binding protein kinase genes in the fruit AZ . Our results demonstrate that in the system of tomato pedicel abscission induced by flower removal KD1 silencing inhibited the upregulation of Ca2+/CaM signaling-related genes. These findings support the previous reports regarding the involvement of Ca2+/CaM signaling in organ abscission, and suggest that KD1 plays a significant role in regulating the Ca2+/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 . 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.  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 .
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 . 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 .
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 . The involvement of auxin and auxin-related gene expression in pedicel abscission of the WT and the KD1-silenced plants was reported previously .
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 . 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 N-terminal (DOMON) domain-containing protein, also called DoH (Fig. 7J), was originally identified in several secreted or cell surface proteins from plants and animals . It is usually associated with other redox domains in larger proteins such as cytochromes b561, and has a suggested capability of transmembrane electron transport [40–42].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.
A second set of genes, which were specifically and transiently upregulated in the WT FAZ plants at 8 h after flower removal, when abscission had already started, were significantly inhibited in the silenced plants (Fig. 8). These data suggest that part of these genes encode proteins that regulate the abscission execution, including: A) Acid Phosphatase (Fig. 8A), whose activity was detected in the AZs of various species, such as sour and sweet cherry fruit, bean leaves, and hibiscus pedicels [43–45]. B) Inositol Hexakisphosphate Kinase3 (Fig. 8B1), to which the inositol hexakisphosphate kinase (InsP6) serves as a cofactor that recognizes auxin and the Aux/IAA polypeptide substrate, Transport Inhibitor Response1 (TIR1) . C) Four Ca+2/CaM-signal transduction regulatory genes, SlCNGC3 (Fig. 8B2), CaM-Like protein (Fig. 9B3), CaM-Binding protein - SlCML35 (Fig. 8B4), and an AAA-ATPase family protein (Fig. 8K2). ATPases Associated with diverse cellular Activities (AAA+-ATPases) are AAA-type ATPase-family proteins, which are involved in cellular functions, such as vesicle transport, organelle assembly, membrane dynamics, and protein unfolding . The Arabidopsis ATPase Family gene 1, (AFG1)-like protein 1 (AFG1L1), which belongs to the extended superfamily of AAA+-ATPase proteins, binds to CaM in a calcium-dependent manner through a CaM-binding site in its catalytic AAA-domain . The function of these genes and the involvement of Ca+2/CaM in abscission were discussed in details above. Our data indicate that different Ca+2/CaM genes regulate early and late events in the abscission process. D) Different transporter-related genes, such as Glycosyltransferase (GTF) (Fig. 8C1), UDP-Glycosyltransferase (Fig. 8C2), three ABC/ABC–2 type transporters (Fig. 8E1-E3), an Amino Acid Transporter (Fig. 9E4), and Vesicular Glutamate Transporter3 (VGULT3) (Fig. 8E5). E) Two decarboxylase genes, AADC1A (Fig. 8F1) and Decarboxylase Family Protein (Fig. 8F2). F) Amino acid biosynthesis/metabolism genes - Glutamine Amido Transferase (GATase) (Fig. 8G1) and WPP Domain-Associated Protein (Fig. 8G2). The Arabidopsis WPP-domain proteins are developmentally associated with the nuclear envelope and promote cell division . G) Cysteine Desulfurase1 (DES1) (Fig. 8H), which encodes the enzyme L-cysteine desulfhydrase that catalyzes the desulfuration of L-cysteine. 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 . 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, 51–54]. 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 , 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, 53–55], is a programmed event, in which these proteins were sequentially increased (Fig. 9).
Phase D of the abscission process, in which the production of a protective defense layer occurs [10, 53–55], was shown to significantly overlap with the execution phase C of abscission . 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.