The Tomato Hybrid Proline-rich Protein regulates the abscission zone competence to respond to ethylene signals

Abstract

The Tomato Hybrid Proline-rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4, significantly inhibited tomato pedicel abscission following flower removal. For understanding the THyPRP role in regulating pedicel abscission, a transcriptomic analysis of the FAZ of THyPRP-silenced plants was performed, using a newly developed AZ-specific tomato microarray chip. Decreased expression of THyPRP in the silenced plants was already observed before abscission induction, resulting in FAZ-specific altered gene expression of transcription factors, epigenetic modifiers, post-translational regulators, and transporters. Our data demonstrate that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: gibberellin perception, Ca²⁺-Calmodulin signaling, Serpins and Small Ubiquitin-related Modifier proteins involved in post-translational modifications, Synthaxin and SNARE-like proteins, which participate in exocytosis, a process necessary for cell separation. These changes, occurring in the silenced plants early after flower removal, inhibited and/or delayed the acquisition of the competence of the FAZ cells to respond to ethylene signaling. Our results suggest that THyPRP acts as a master regulator of flower abscission in tomato, predominantly by playing a role in the regulation of the FAZ cell competence to respond to ethylene signals.

Fig. 1. Effect of antisense silencing of THyPRP (ID—X57076/Solyc07g043000) on the kinetics of changes in qPCR expression of THyPRP in the FAZ at various time points after flower removal.

qRT-PCR analysis of THyPRP expression in the FAZ of antisense transgenic line 11/generation T4 was compared to that in the WT at 0, 4, 8, 12, 16, and 20 h after flower removal. The relative quantification of the gene expression level in the qPCR assay was determined by the comparative C_T method 2^-∆∆CT, using ACTIN as the reference gene. The data represent the mean values (±SE) of duplicate experiments, each having three independent biological samples

Fig. 2. Effect of antisense silencing of THyPRP on the kinetics of flower pedicel abscission following flower removal.

Flower explants were prepared and handled as previously described^,. Wild type (WT) plants (cv. NY) and silenced lines 7 and 11/generation T4 were used. The percentage of accumulated pedicel abscission was monitored at 0, 4, 8, 12, 16, and 20 h after flower removal. The results are means of four replicates (n = 30 explants) ± SE

Fig. 3. Effect of antisense silencing of THyPRP on the kinetics of changes in array-measured expression levels of genes related to the plant hormones.

Ethylene (a–c), Auxin (d, e), and Gibberellin (f, g), and Polyamine (h), that were specifically upregulated in the WT FAZ at various time points after flower removal: early-4 h (blue*) or 8 h (red*), or late 12–20 h (green*).TAPG4::antisense THyPRP-silenced line 11/generation T4 was used. Expression levels were measured for Blue copper-like protein1 (BCP1) (a); 1-Aminocyclopropane-1-Carboxylate Synthase (ACS) (b1); 1-Aminocyclo-propane-1-Carboxylate Oxidase (ACO) genes (b2–b5); Ethylene-Related Factor (ERF) TF genes (c1–c10); Small Auxin-Up RNA (SAUR) genes (d1, d2); Gretchen Hagen3 (GH3) which are Indole-3-acetic acid-amido synthetase genes (e1, e2); GA receptor (GID1L2) (f); GA-regulated protein (GASA) genes (g1–g4); and Ornithine Decarboxylase (ODC) (h). Transcript identities are indicated by their gene ID and their Arabidopsis (At) gene number and/or their nucleotide accession number. The results are means of two independent biological replicates ± SD

Fig. 4. Effect of antisense silencing of THyPRP on the kinetics of changes in array-measured expression levels of genes related to transcription factors (TFs) that were specifically upregulated in the WT FAZ at various time points after flower removal: early—4 h (blue*) or 8 h (red*), or late 12–20 h (green*).

TAPG4::antisense THyPRP-silenced line 11/generation T4 was used. Expression levels were measured for tomato MYB TF genes (a1–a5); Zink finger (Znf) TF genes (b1–b11); Basic helix-loop-helix (bHLH) TF genes (c1–c3); Basic leucine zipper (bZIP) (d); GATA TF (e); Ring finger TF genes (f1, f2); and WRKY TF genes (g1–g5). Transcript identities are indicated in the graphs by their gene ID and their Arabidopsis (At) gene number and/or their nucleotide accession number. The results are means of two independent biological replicates ± SD

Fig. 5. Line graphs showing the kinetics of changes in array-measured expression levels of genes that were specifically and continuously downregulated [I] or upregulated [II] at zero time and later on, or upregulated at 4 (blue*) or 8 (red*) h after flower removal [III] in the FAZ of THyPRP-silenced plants.

TAPG4::antisense THyPRP-silenced line 11/generation T4 was used. Expression levels were measured for tomato Long-chain fatty acyl-CoA synthetase (IA); Lipid transfer protein (IB); High Mobility Group (HMG) type nucleosome factor (IC); MKIAA0930 (ID); Serine protease inhibitor (Serpin) (IIA); Peptide transporter (IIB); Nucleotide Binding Site—Leucine-Rich Repeat (Cc-NBS-LRR) (IIC); Ubiquitin-like protein1 (Ulp1 protease) (IID); unknown proteins (IIE1–E6); FAD-binding domain-containing protein (IIF); WUSCHEL-related homeobox-containing protein4 (WUS) (IIG); SlbHLH transcription factor148 (IIH); HTC in fruit (II–I); HAT dimerization domain-containing protein (IIJ); Nitrate transporter-TGF (IIK); MtN3-like protein (IIL); DUF599 family protein (IIM); Myrosinase-Binding protein2 (MBP2) (IIN); Plant cell wall protein SlTFR88 (IIIA); Uridine 5′-diphospho (UDP)-glucuronosyltransferase (IIIB); Transcription factor HBP-1b(c1)-like (IIIC); CONSTANS-like ZF (IIID); CONSTANS1 (CO1) TF (IIIE); Rhamnogalacturonate endolyase (IIIF); Disease resistance protein (IIIG); and defense-related Receptor-Like protein Kinase (RLK) (IIIH). Transcript identities are indicated in the graphs by their gene ID and their Arabidopsis (At) gene number and/or their nucleotide accession number. The results are means of two independent biological replicates ± SD

Fig. 6. Effect of antisense silencing of THyPRP on the kinetics of changes in array-measured expression levels of genes that were specifically upregulated in the WT FAZ at various time points after flower removal: early—4 h (blue*) or 8 h (red*) or late 12–20 h (green*).

TAPG4::antisense THyPRP silenced line 11/ generation T4 was used. Expression levels were measured for protein degradation–ubiquitin-related genes—F-box or U-box (a1–a5); genes related to transporters of macromolecules—C2 domain-containing protein (b1) and Syntaxin (b2); ABC transporter genes (c1–c3); Cyclic nucleotide-gated ion channels (c4); ATPase-AAA cell division (d); Calcium-transporting ATPase1 (e1); Calmodulin-binding protein genes (e2, e3); Calmodulin-like protein genes (e4–e5); Prephenate dehydrogenase hydrolyase (f); Lateral organ boundaries (LOB) lipids and wax-related genes—photoassimilate-responsive-1c (PAR-1c protein) (g1); LOB-domain protein25 (g2); Non-specific lipid-transfer protein (g3); Diacylglycerol kinase1 (DGK1) (g4); Fatty acid desaturase (g5); Lipoxygenase (g6); receptor kinase and protein phosphatase-related genes—Serine/threonine-protein kinase receptor (h1); Receptor-Like protein Kinase (RLK) (h2); Phosphoenolpyruvate carboxykinase (h3); Serine/threonine-protein phosphatase7 (h4); Serine/threonine-Protein Phosphatase6 regulatory Ankyrin Repeat Subunit A (PP6-ARS-A) (h5); Redox regulation genes—Glutaredoxin (i1); Tropinone reductase1 (i2); and Ribosomal-protein-alanine N-acetyl-transferase (j). Transcript identities are indicated in the graphs by their gene ID and their Arabidopsis (At) gene number, and/or their nucleotide accession number. The results are means of two biological independent replicates ± SD

Fig. 7. Summary of the genes modified in the FAZ of TAPG4::antisense THyPRP plants before (at zero time) and at 4 h after flower removal in response to THyPRP downregulation and abscission induction by flower removal, thereby leading to decrease of the postulated events resulting in the inhibition of pedicel abscission.

Genes that were specifically upregulated or downregulated in the FAZ of THyPRP-silenced plants compared to the WT are marked in Green or Red, respectively. The numbers listed besides the gene names indicate the number of genes in the gene family that were affected in THyPRP-silenced plants