Auxin conjugation and hydrolysis play important roles in mediating the auxin balance for proper plant development and response to environment signals [2, 23]. Several auxin conjugate hydrolases have been isolated from the dicots Brassica rapa, Medicago truncatula, and Arabidopsis thaliana and the monocots Triticum aestivum and rice, and are involved in root development, pathogenesis, and grain weight [9, 16, 24,25,26]. The biochemical characterization of substrate specificity and the different temporal and spatial gene expression patterns further revealed the complex role of IAA conjugate hydrolases in auxin signaling [25, 27]. Although our understanding of IAA-regulated abscission processes, including the molecular mechanisms of action, perception, and transport, comes from studies of gene transcription analyzed in pre-abscission or auxin-depleted induced abscission, there remains a lack of knowledge about the potential role of IAA conjugate hydrolases in mediating abscission [20, 22, 28].
Identification, sequencing, and catalytic activity analysis of ILL genes and proteins in tomato
In Arabidopsis, seven genes of the amidohydrolase family have been well characterized: ILR1, ILL1, ILL2, and IAR3 can cleave IAA amino acid conjugates, while ILL3 and ILL6 cannot, and ILL5 is thought to be a pseudo gene [11, 26, 29]. Genome scanning and DNA sequencing identified seven full-length tomato cDNAs, SlILL1 through SlILL7, that are highly homologous to the IAA-amidohydrolases of Arabidopsis. The IAR3-like (Arabidopsis and Brassica) AtILL1 and AtILL5 proteins contain a C-terminal KDEL sequence that signals retention of plant proteins in the lumen of the ER. However, none of the SlILL proteins contains a terminal KDEL, and are therefore predicted to have a lower probability of being located on the ER, although SlILL1, 2, and 7 have a terminal HDEL motif (similar to Br-ILL6), which is annotated by PSORT as an ER retention signal . The importance of ER localization is not clear, however, because phylogenetic analysis of 66 orthologs from across the plant kingdom resolved two separate monocot clades, one with members possessing ER retrieval sequences and one lacking them [2, 30, 31]. The ER retention signal is not required for activity, because two of the five amidohydrolases characterized from M. truncatula lack such a tetrapeptide, but still show activity , and TaIAR3 has the unusual C-terminal sequence motif RDEL . Moreover, a cleavable N-terminal signal sequence was found in SlILL1, 2, 3, 4, and 7, while SlILL5 and 6 contain an uncleavable N-terminal sequence.
Previously, the structure of AtILL2 was found to be similar to members of the M20 peptidase family, which suggests that ILL2 likely uses a catalytic mechanism similar to that established for the M20 peptidase enzymes family. All SlILLs have the M20 dimerization domain, which implies that all of them have auxin conjugate hydrolysis activity. The experiment further indicated that all SlILLs are able to hydrolyze IAA-Ala. The different enzymes also showed distinct patterns of hydrolase activity. AtILL2 preferentially catalyzes the hydrolysis of IAA-Ala and the hydrolytic activity is higher than that of SlILLs, while AtILL1, which shares 87% sequence identity with AtILL2, showed a lower activity than the SlILLs toward IAA-Ala. AtILR1 prefers IAA-Leu, IAA-Tyr, and IAA-Phe, while showing less activity toward IAA-Ile, and less activity than SlILLs. ILL3 and ILL6/GR1 have no such catalytic activity towards IAA conjugates [11, 30]. In Triticum aestivum, TaIAR3 has far less activity than SlILLs toward all of IAA-Ala, IAA-Ile, IAA-Asp and IAA-Gly substrates . In Medicago truncatula, MtIAR31, − 32, − 33, and − 34 have hydrolytic activity toward IAA-aspartate and IBA-alanine, and shows higher activity than SlILLs toward IAA-Asp . No hydrolase activity was found for IAA–Asp in vitro for all seven SlILLs; IAA–Asp may be an uncleavable auxin conjugate, and has no effect on abscission, hence it is possible that there is no specific auxin conjugate hydrolase for IAA-Asp in tomato.
Auxin conjugates are involved in flower pedicel abscission
Cellular IAA is mainly present as amide derivatives and, to a lesser extent, as ester-linked conjugates, in Arabidopsis thaliana . The most common auxin conjugates in Arabidopsis are IAA-Ala, IAA-Leu, IAA-Asp, IAA-Glu, IAA-glucose, and protein/peptide conjugates [23, 32,33,34,35]. Auxin conjugation is reversible through the actions of specific hydrolases, with the subsequent release of free IAA. Therefore, IAA conjugates have auxin activity when applied exogenously and show physiological activity in regulating different developmental processes, such as seed germination and root elongation.
The level of endogenous auxin must fall below a certain threshold in the abscission zone to initiate abscission [36, 37]. Endogenous auxin would be synthesized de novo and is mainly derived from young organs such as flowers, young fruits, and seeds; therefore, the local auxin concentration in the AZ is mainly mediated by auxin flux from flowers to stems. Flower removal abolishes auxin influx and then activates conjugate hydrolyase enzymes to release active IAA; however, this is insufficient to keep the AZ insensitive to ethylene and prevent abscission, which usually only lags the initial abscission by several hours. For complete inhibition of abscission, exogenous auxin is required. In this study, abundant IAA-Ala and IAA-Ile could completely inhibit abscission, while IAA-Asp had little effect. As previously reported, IAA-Ala possesses auxin activity without hydrolysis, so we chose to use IAA-Ile to study the effects of an auxin conjugate on abscission. IAA–Asp is thought to be involved in a degradation pathway and, because it does not release free auxin, it has a minimal role in abscission. In auxin-depleted induced tomato pedicel abscission, IAA in the AZ declined during the early stages (before 8 h) when abscission was initialized, then increased at 16 h, which is an ethylene-accelerated abscission stage. About 60% percent of the flowers abscised during this stage. The low auxin concentration in the AZ is linked to increased ethylene sensitivity, and might also result in secondary cell wall deposition at the site of the reduced auxin response, which is instrumental during the separation process [38, 39]. The different times of treatment showed that before 6 h, IAA-Ile could effectively delay abscission, while there was little effect after 8 h. Moreover, incubation with IAA-Ile for 2 h was sufficient to inhibit abscission. These results showed that there is not enough auxin conjugate in the AZ to inhibit abscission, and that the optimal time to inhibit abscission by exogenous auxin conjugate application is before 6 h.
To determine whether de novo synthesized auxin conjugates are required for abscission, the tomato pedicel explants were incubated in MS agar for different times then transferred to IAA-Ile medium containing 10 mM CHX (cycloheximide). Inhibition of de novo synthesis of auxin conjugate hydrolase enzymes would decrease the auxin concentration and accelerate abscission. Our results showed that induced auxin conjugate hydrolases are required for the release of more free auxin to delay abscission. The results also indicated that there is not enough auxin conjugate hydrolase activity before 4 h and insufficient levels of auxin conjugate after 8 h. In our previously study, the early abscission stage was found before 8 h, and the dynamics of free auxin in the AZ seems to be a major regulator for this process .
Expression of tomato SlILL genes during abscission
Assaying SlILL gene expression in the different tomato organs showed that SlILL1, 3, 4, and 7 are expressed at a relatively low levels, while the expression of SlILL2 and 5 is somewhat higher in the AZ compared with that in other organs. Notably, only SlILL6 had a high level of expression in the AZ. Moreover, SlILL1, 5, and 6 showed unique expression patterns in the AZ, and the expression of all three genes was altered by 1-MCP and auxin treatments, showing that gene expression is highly associated with abscission. A previous report showed that wounding could induce the expression of auxin conjugate hydrolases, such as IAR3 in Arabidopsis, and a nonspecific increase in expression of both SlILL3 and 7 in the AZ and NAZ might be a manifestation of the wound response. However, in another view, SlILL1, 3, 5, 6, and 7 all showed a trend of increasing expression during abscission in the AZ, which may not exclude their potential role in releasing auxin to delay abscission. This also draws attention to the fact that expression of all SlILL genes is inhibited under auxin treatment. The expression of SlILL genes is mediated by free auxin, which might be required for precise auxin signaling in mediating AZ abscission. In addition, the auxin conjugate hydrolase enzymes are regulated by the free auxin content at the transcriptional level, which might be important to determine the precise auxin concentration required for cell development and for responses to environmental stimuli.
Transcription and translation of tomato SlILLs protein during abscission
SlILL1, 5, and 6 showed special expression profiles during abscission; therefore, they were selected as abscission-related auxin conjugate hydrolase genes for western blotting analysis. The pattern of SlILL1, SlILL5 and SlILL6 protein levels in response to 1-MCP and IAA indicated that they were possibly related to abscission.
SlILL1, 5, and 6 are major regulators of delayed abscission
The plants in which single SlILL genes were silenced by VIGS showed a normal phenotype, but had reduced sensitivity to specific auxin conjugates. The more hydrolase genes that were downregulated, the less sensitive the plants became to exogenous auxin conjugates [11, 40]. Double and triple mutants of Arabidopsis auxin conjugate hydrolases showed lower auxin levels in seedlings and defective responses to exogenous auxin conjugates [11, 26]. In this study, all tomato hydrolases showed distinct expression patterns and cleavage activities, implying that they might have distinct, yet overlapping, functions during abscission. Silencing SlILL1, SlILL5, and SlILL6 individually resulted in significantly accelerated abscission in pedicel explants, while double silencing showed that SlILL3 and 7 had little effect on abscission. The double-silenced SlILL1 + 5, SlILL1 + 6, and SlILL5 + 6 explants showed a significant increase in abscission compared with that of the single gene-silenced explants, implying that SlILL1, 5, and 6 play important roles in the abscission process. The triple-silenced lines further supported this hypothesis, since they showed a further accelerated abscission rate compared to the double-silenced VIGS lines. Taken together, these results indicated that the auxin conjugate hydrolase enzymes encoded by SlILL1, SlILL5, and SlILL6 coordinate to mediate pedicel abscission in tomato. We also used VIGS1 + 5 + 6 lines to investigate restoring the phenotype, The result showed that IAA addition could restore the phenotype of the VIGS lines (Additional file 5: Figure S3).
The concept that the AZ local auxin concentration plays a negative role in mediating abscission procession has been understood for 50 years. Artificial activation of the bacterial auxin biosynthetic genes iaaL and iaaM in the AZ further supports this view . Moreover, auxin is transported in a polar manner to other organs mediated by PIN efflux carriers that determine the direction of flux . It is also important to prevent abscission by maintaining this constant auxin flux . The effect of auxin on PIN expression has been well- studied. Below the optimal concentration, the auxin-induced PIN expression effect was correlates positively with the amount of auxin, while PIN expression was reduced when the auxin concentration was far in excess of the optimal concentration. In AZ, the auxin concentration is relatively low, and depressed SlILR1, 5, and 6 expression in the triple silenced line showed further downregulation of the AZ auxin concentration, leading to a low expression of PIN3, 4, and 8, which might decrease auxin polar transport . The mechanism by which SlILL1, SlILL5, and SlILL6 mediate abscission, not only via altered auxin content, also via auxin flux by affecting PIN gene expression. It is reasonable to propose that the local auxin content in the AZ and auxin transport are tightly connected to regulate abscission.