**3. Transduction of SL signal**

that serine from the catalytic triad is involved in docking of SLs into D14 [38]. With an aver‐ age rate of 0.3 molecule/min, D14‐mediated hydrolysis of SLs into non‐active derivatives is very slow, indicating that this is not the main function of D14 [33, 36]. Crystallographic analysis indicates that the degradation of SL molecules by D14 brings about a change in receptor conformation, which is necessary for the interaction between D14 and other com‐ ponents from SL‐signaling pathway [39]. After nucleophilic attack and release of the ABC part, the D ring remains within the receptor that now assumes a "closed" state unable to bind further molecules, reviewed by Waters [7]. This change in conformation destabilizes the D14 receptor, thus initiating its own degradation [40] (**Figure 5**). This is the first known case where hormone hydrolysis by a receptor causes the degradation receptor as well.

106 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

Since SLs are involved in the regulation of the development of different organs, it was expected that D14 will be located in almost all plant tissues. Expression analysis of *Atd14* in *A. thaliana*, however, showed markedly higher levels in vascular tissues of roots and shoots [41]. The discrepancy between expression and distribution pattern of the D14 protein was explained, when the intercellular transport of D14 *via* the phloem was uncovered. This transport is SL‐independent and also in the SL biosynthesis mutants it was observed that D14 was delivered into axillary buds [42] leaving the question by what mechanism D14 is transported and how it is been used by plants to regulate the development and adaptation

**Figure 5.** Overview of SL‐signaling cascade including hydrolysis of SL molecules by receptor and change of the receptor conformation, which allows the interaction with the SCF complex and repressor. Ubiquitination of the repressor,

mediated by the SCF complex, results in the expression of genes from the TCP family.

to different stresses.

The common mechanism for transducing phytohormone signals is the degradation of pro‐ teins called repressors. This degradation is mediated by a SKP1‐Cullin‐F‐box complex (SCF), composed of three proteins that upon binding to repressors mark them for proteasomal deg‐ radation *via* ubiquitination. Cullin is a main structural part of the SCF complex controlling the connection of the whole complex to the ligase E3. The SKP1 component is responsible for the binding of the specific F‐box protein which designates the protein for degradation [43]. In theory, the F‐box protein renders specificity to the whole CSF complex and should be specific for different hormones. In practice, however, evidence shows that other components interact‐ ing with the SCF complex can influence its specificity.

An F‐box protein which was part of an SCF complex and involved in SL signaling was identi‐ fied in the *A. thaliana*‐mutant *max2* and the rice‐mutant *d3* that were insensitive to treatment with SLs [44, 45]. For the long time that mutants were used to describe the role of SLs in the plant growth and development, recently MAX2/D3 has been found to be involved in both SL‐ and KAR‐signaling pathways, reviewed by Waters [7]. For this reason, the phenotype of *max2*/*d3* mutants cannot be directly linked to the function of SLs. Evidence showing that MAX2‐guided degradation of transcription factors that is dependent also on the other phyto‐ hormone class of brassinosteroids (BRs) indicates that this F‐box protein fulfills a wide range of this F‐box protein in hormone signaling [46].

In *A. thaliana,* MAX2 interacts with AtCullin1 and Arabidopsis Serine/Threonine Kinase1 (ASK1) [47], whereas in rice D3 is part of a SCF complex together with OsCullin1 and *O. sativa* SKP1‐Like 1/5/20 (OSK 1/5/20) [48]. Although these complexes indicate a conserved mechanism for SL signal transduction in both mono‐ and dicots, the presence of three differ‐ ent OSKs in rice may suggest that complexes with OSK1/5/20 recognize different substrates and are involved in different SL‐dependent processes [48]. MAX2/D3 show nuclear localiza‐ tions and SL‐dependent interactions between F‐box protein D3 with SL receptor D14 were reported in rice. Obtained results indicate that this interaction is mediated by the presence of SLs, and it depends on the concentration of SLs and it is also stereoisomer‐specific. In a "closed" state, D14 is able to interact with D3 (**Figure 5**), while the version of D14 with muta‐ tions in or near the active pocket site shown reduced interactions with D3. This suggests that after SL‐mediated changed conformation of D14, D3 can bind D14 close to the active pocket side entry [40].

Based on the similarity of MAX2/D3 protein to other hormone receptors such as jasmonate receptor Coronatine Insensitive1 (COI1) [49] or auxin receptor Transport Inhibitor Response1 (TIR1) [50], it was predicted that MAX2/D3 may also be involved in SL perception. Although there is no evidence that MAX2/D3 can interact with SLs, there are reasons to assume that MAX2/D3 may act as a receptor for other signaling molecules.

For a long time, it was not known which proteins are recognized by the SCFMAX2/D3 complex, but recently the SL repressors degraded during SL signal transduction were identified in rice (D53) [51, 52] and in *A. thaliana* (Suppressor of MAX2‐Like 6 to 8, SMXL 6 to 8) [53–55]. Gain‐of‐function mutation in D53 resulted in semi‐dwarf plants with increased tillering, a phenotype which is characteristic for SL mutants. Similar effects were observed after over‐ expression of *D53*, whereas reduced expression of *D53* in a *d53*‐mutant background inhib‐ ited tiller formation [52]. D53 shows the nuclear localization and it was confirmed that in the presence of SL molecules degradation of D53 occurs. This degradation proceeds through the proteasome‐dependent pathway and requires the presence of D14 and D3 proteins [51, 52]. There are evidences that D53 may also interact with D3 in the absence of D14, although this interaction is less efficient. In contrast to rice, where only one SL repressor has been identi‐ fied, *A. thaliana* contains three proteins—SMXL 6 to 8—that may act redundantly. First reports indicated that only a triple mutant *smxl6/7/8* will result in a phenotype with reduced number of tillers [54]. Later on, it was found that the phenotype characteristic for SL mutants could be produced by the expression of the non‐degradable form of SMXL 7 under a native promoter [55]. All three SMXLs interact with D14 and are degraded in an SL‐dependent manner in the presence of MAX2 and D14 [53, 54]. It still remains an open question whether SMXLs 6 to 8 do act redundantly or they are involved in different responses to SLs.

The SL repressors of both *A. thaliana* and rice contain the conserved amino acid sequences (F/L‐D‐L‐N‐L) which is known as an ethylene‐responsive element‐binding factor‐associated amphiphilic repression (EAR) motif. This motif plays a key role in interactions with transcrip‐ tional corepressors from Topless (TPL) and Topless‐Related Proteins (TPR) families [52, 53]. TPL and TPR regulate the expression of genes in response to different classes of hormones, such as auxin or jasmonates [56]. The presence of an EAR motif in SL repressors suggests that D53/SMXLs may bind TPL/TPR corepressors. An ensuing degradation of these corepressors may then result in the expression of transcriptional factors, previously suppressed by TPL/ TRP. Interactions between SMXL6 to 8 and proteins from the TPR family were already con‐ firmed using a yeast‐two hybrid and co‐immunoprecipitation assays [54].
