A tetraspanin gene regulating auxin response and affecting orchid perianth size and various plant developmental processes

Abstract The competition between L (lip) and SP (sepal/petal) complexes in P‐code model determines the identity of complex perianth patterns in orchids. Orchid tetraspanin gene Auxin Activation Factor (AAF) orthologs, whose expression strongly correlated with the expansion and size of the perianth after P code established, were identified. Virus‐induced gene silencing (VIGS) of OAGL6‐2 in L complex resulted in smaller lips and the down‐regulation of Oncidium OnAAF. VIGS of PeMADS9 in L complex resulted in the enlarged lips and up‐regulation of Phalaenopsis PaAAF. Furthermore, the larger size of Phalaenopsis variety flowers was associated with higher PaAAF expression, larger and more cells in the perianth. Thus, a rule is established that whenever bigger perianth organs are made in orchids, higher OnAAF/PaAAF expression is observed after their identities are determined by P‐code complexes. Ectopic expression Arabidopsis AtAAF significantly increased the size of flower organs by promoting cell expansion in transgenic Arabidopsis due to the enhancement of the efficiency of the auxin response and the subsequent suppression of the jasmonic acid (JA) biosynthesis genes (DAD1/OPR3) and BIGPETAL gene during late flower development. In addition, auxin‐controlled phenotypes, such as indehiscent anthers, enhanced drought tolerance, and increased lateral root formation, were also observed in 35S::AtAAF plants. Furthermore, 35S::AtAAF root tips maintained gravitropism during auxin treatment. In contrast, the opposite phenotype was observed in palmitoylation‐deficient AtAAF mutants. Our data demonstrate an interaction between the tetraspanin AAF and auxin/JA that regulates the size of flower organs and impacts various developmental processes.

of lips and sepals/petals in orchids is controlled by L (lip) and SP (sepal/petal) complexes, respectively (Hsu et al., 2015). The higherorder heterotetrameric L (lip) complex is exclusively required for lip determination, while the SP (sepal/petal) complex specifies sepal/ petal formation (Hsu et al., 2015). How exactly orchid perianth formation and characteristics, such as morphological features and size, are regulated after P-code complexes are established remains obscure. For example, lips are much larger than sepals/petals in Oncidium orchids. By contrast, lips are much smaller than sepals/ petals in Phalaenopsis orchids. It is therefore a reasonable assumption that these two orchid genera have opposing mechanisms for regulating perianth size in the lips and sepals/petals. TETRASPANIN genes have been identified in multicellular eukaryotes (Huang et al., 2005;Lambou et al., 2008;Wang et al., 2015) but were absent in yeast (Garcia-España et al., 2008). The structures of tetraspanin family proteins were evolutionary conserved (Zuidscherwoude et al., 2015) and contained five distinct regions, including a large extracellular domain, a small extracellular domain, transmembrane domains, palmitoylation sites, and cytoplasmic domains (Stipp, Kolesnikova, & Hemler, 2003). In mammalian cells, tetraspanins form a tetraspanin-enriched microdomain (TEM), known as a tetraspanin web, by interacting with one another, specific lipids and other transmembrane proteins, including integrins and other adhesion receptors (Charrin, Jouannet, Boucheix, & Rubinstein, 2014;Hemler, 2005;Reimann, Kost, & Dettmer, 2017;Zuidscherwoude et al., 2015). The organization of the integrin-tetraspanin microdomain and modulation of adhesion-dependent signaling were mediated by a posttranslation modification of tetraspanins via palmitoylation (Berditchevski, Odintsova, Sawada, & Gilbert, 2002). The mutation of juxtamembrane cysteines, the palmitoylation site of a human tetraspanin CD81, reduced the ability of CD81 to interact with other proteins (Delandre, Penabaz, Passarelli, Chapes, & Clem, 2009).
TET5 and TET6 have redundant functions in restricting cell proliferation during root and leaf growth (Wang et al., 2015). TET13 has a function in the primary root, affecting apical meristem size and root length, and in lateral root initiation (Wang et al., 2015). However, the functions of most plant TETRASPANIN genes still remain to be investigated (Reimann et al., 2017).
When roots are under 90-degree gravitropic stimulation, auxin redistributes to the lower side of the roots and inhibits the elongation of epidermal cells, causing root bending (Sato, Hijazi, Bennett, Vissenberg, & Swarup, 2015). In addition, lateral root primordia are triggered by gravitropic stimulus and develop in eight stages (Péret et al., 2012).
Auxin has been thought to regulate petal development since the disrupted expression of an auxin-inducible indicator DR5 were observed in petal loss (PTL) mutant plants (Lampugnani, Kilinc, & Smyth, 2013). The sizes of petals and the lengths of stamens were significantly reduced in Arabidopsis auxin response factor 6 (arf6) and arf8 double-mutant plants (Nagpal et al., 2005). Auxin may regulate petal development and anther dehiscence through the regulation of jasmonate (JA) activity, since it has been shown that exogenous auxin inhibits the expressions of the JA biosynthesis gene DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and 12-oxophytodienoate reductase (OPR3) and results in anther indehiscence (Cecchetti et al., 2013).
Interestingly, petal size is increased in opr3 mutants due to larger cell sizes at developmental stage 14 instead of stage 9, indicating that JA controls petal size by suppressing cell expansion at late stages (Brioudes et al., 2009). This assumption was supported by the fact that JA regulates an alternative splicing event in the ubiquitously expressed gene BIGPETAL (BPE) to translate a basic helix-loop-helix (bHLH) transcription factor BPEp that inhibits cell expansion in late development stages of petal growth (Brioudes et al., 2009;Varaud et al., 2011).
Auxin is also involved in response to abiotic stresses such as drought, salt, and cold. Exogenous auxin enhanced tolerance to drought (Shi et al., 2014) and rescued the salt hypersensitivity phenotype of plants overexpressing TTG2/WRKY44 .
In this study, we identified the tetraspanin gene Auxin Activation Factor (AAF) from Oncidium and Phalaenopsis orchids and demonstrated that the expression of orchid AAF orthologs is associated with the regulation of the perianth size after its identity is determined by the P-code complexes. Further analysis of an Arabidopsis AAF gene reveals that AAF controls not only flower organ size but also various developmental processes such as anther dehiscence, drought tolerance, and lateral root formation by enhancing the efficiency of the auxin response in plants. Our results suggest a novel interaction between the tetraspanin gene AAF and auxin in regulating plant growth and development.

| Plant materials and growth conditions
Seeds for Arabidopsis were sterilized and placed on agar plates containing Murashige and Skoog medium (Murashige & Skoog, 1962) at 4°C for 2 days. The seedlings were then grown in growth chambers under long-day conditions (16-hr light/8-hr dark) at 22°C for 10 days before being transplanted to soil. The light intensity of the growth chambers was 150 μE m −2 s −1 . Species, cultivars, and peloric mutants of orchids used in this study, including the Oncidium (O. Lemon Heart and the associated peloric mutants O. Lemon Heart Trilips) and moth orchids (Phalaenopsis Sogo Yukidian "V3," P. Red Bell, P. Gold Diamond, and the associated peloric mutants P. Big-Lip), were maintained in the greenhouse of National Chung-Hsing University, Taichung, Taiwan.

| Cloning of orchid AAF cDNAs
The transcriptomic RNA-Seq for Oncidium (O. Lemon Heart) and Phalaenopsis (P. Sogo Yukidian "V3") floral organs (lip and sepal/petal) at floral bud stage (6 mm for Oncidium and 15 mm for Phalaenopsis) was performed and data analyzed. An Oncidium Auxin Activation Factor (OnAAF) gene which expressed specifically higher in lip than in sepal/petal of Oncidium was identified. PaAAF which showed highest sequence identity/similarity to OnAAF and expressed specifically higher in petal than in lip of Phalaenopsis was also identified.
The cDNA contained the 3′-end of OnAAF was obtained by 3′-RACE using the BD SMART RACE cDNA Amplification Kit (Clontech Laboratories) following the 5′ gene-specific primer OnAAF-1. The full-length cDNA of OnAAF was amplified by PCR using 5′ primer, OnAAF-1, and the 3′ primer, OnAAF-2. The full-length cDNA of Phalaenopsis PaAAF was amplified by PCR using 5′ primer, PaAAF-1, and the 3′ primer, PaAAF-2. Sequences for the primers are listed in Table S1.  Table S1.

| AtAAF::GUS fusion construct
For the AtAAF::GUS construct, the AtAAF promoter (2.0 kb) was obtained by PCR amplification from the genomic DNA using the pMSIF-1 and pMSIF-2 primers and then cloned into pGEM-T easy vector (Promega). The full-length promoter for AtAAF (2.0 kb) was then subcloned into the linker region before the β-Glucuronidase (GUS) coding region in binary vector pEpyon-01K (CHY Lab). The primers contained the generated PstI (5′-CTGCAG-3′) recognition site and SalI (5′-GTCGAC-3′) recognition site to facilitate the cloning of the promoter. Sequences for the primers are listed in Table S1.

| Construction of AtAAF+GFP construct
AtAAF cDNAs were subcloned into the multiple cloning site of binary vector pEpyon-12K (CHY Lab) upstream of the mGFP5 sequence and under the control of the CaMV 35S promoter. The fragments contained the generated XbaI and KpnI recognition site to facilitate the cloning of the AtAAF. This construct was used for plant transformation. The sequences for the primers are listed in Table S1.

| Real-time PCR analysis
For real-time quantitative RT-PCR, the reaction was performed on an  Table S1. The Arabidopsis housekeeping gene UBQ10 was used as a normalization control with the following primers: RT-UBQ10-1 and RT-UBQ10-2.
The transcript levels for orchid genes were determined using three replicates and were normalized using reference genes ACTIN (primers: RT-PACT4-1 and RT-PACT4-F) for Phalaenopsis (Hsu et al., 2015) and α-tubulin (primers: RT-OTUB-1 and RT-OTUB-2) for Oncidium (Chang et al., 2010) as described previously (Hsu et al., 2015). The data were analyzed using CFX Manager™ software (version 3.1; Bio-Rad) according to the manufacturer's instructions. The "delta-delta method" for- △CP control] , where represents perfect PCR efficiency, was used to calculate the relative expression of the genes. To calculate the statistical significance, unpaired t tests were used.

| Plant transformation and transgenic plant analysis
Constructs made in this study were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis plants using the floral dip method as described elsewhere (Clough & Bent, 1998). Transformants that survived in the medium containing kanamycin (50 μg/ml) were further verified by RT-PCR analysis.

| Stress treatments
For drought treatment, 1-week-old Arabidopsis seedling was removed from the MS medium and exposed to a stream of air for various times. Subsequently, seedlings were weighted, the pictures of seedlings were taken and the RNA of seedlings was extracted for gene expression analysis. For salt stress treatment, 1-week-old seedlings were removed from the MS medium and NaCl were added to the liquid MS medium to a final concentration of 150 mM. Seedlings were dipped for various times, and the RNA of seedlings was extracted for gene expression analysis. Plants were grown in MS medium plate with 150 mM NaCl for survival rate calculation.

| Alexander's staining
For pollen analysis, the pollen grains were mounted with Alexander's stain as previously described (Alexander, 1969).

| Cryo-scanning electron microscopy
Cryo-scanning electron microscopy (SEM) was performed according to the method as previously described (Hsu et al., 2015).

| Confocal laser scanning microscopy
The flower tissues were imaged using an Olympus FV1000 confocal microscope as described previously (Chang et al., 2010;Peng et al., 2013). The plant cell walls were stained with 40 μg/ml propidium iodide (PI; Molecular Probes). PI was excited by a 543-nm He/Ne laser line, and the emission was collected at 555-655 nm.

| Application of jasmonate
All opened flowers (after stage 14) were removed from the inflorescence, and the remaining flower bud clusters were dipped into 50 µM (±) Jasmonate (Sigma) dissolved in 0.05% aqueous Tween 20.

| Measurement of the concentration of the IAA
Flower buds of Arabidopsis were ground under liquid nitrate then resuspended in 90 μl phosphate-buffered saline (PBS) buffer for total IAA extraction. The IAA quantification was performed by enzymelinked immunosorbent assay (ELISA) kit (LYBDBio).

| Biotin switch assay of S-acylation
The biotin switch assay was performed as previously described (Hemsley, Taylor, & Grierson, 2008)  Hydroxylamine was replaced by Tris-HCl buffer in the remaining aliquot as a control. Free reagent was removed as mentioned above, and 12 μl of the solution was removed as a loading control.
Biotinylated proteins were then purified with 15 μl NeutrAvidin-agarose (Thermo Fisher Scientific) and analyzed by Western blotting using GFP-specific antibodies.

| Virus-induced gene silencing experiment
The virus-induced gene silencing (VIGS) experiments in orchids were performed as described previously (Hsu et al., 2015).

| The expression of Oncidium OnAAF is positively correlated with the growth of lip and is down-regulated in OAGL6-2 VIGS small lips
To explore how orchid perianth formation and characteristics, The Oncidium Auxin Activation Factor (OnAAF) is a member of the tetraspanin family in the TET7/8/9 group (Figures S1 and S2) encoding a protein of 270 amino acids ( Figure S1) which showed the 57% identity and 75% similarity to its Arabidopsis TETRASPANIN orthologue AtAAF (At4g30430) (originally known as TET9) (Figures S1 and S2). The OnAAF protein contained four transmembrane domains, two extracellular loops, and cytoplasmic N and C terminals ( Figure S1). When the sequence of the OnAAF protein was further analyzed, four palmitoylation sites were predicted by CSS-Palm (Zhou, Xue, Yao, & Xu, 2006) ( Figure S1).
In the Oncidium Trilips mutant (Figure 1c), OnAAF was clearly upregulated in the enlarged lip-like petals (Figure 1d), indicating that OnAAF likely promoted lip expansion after the P code was established

| The expression of Phalaenopsis PaAAF is positively correlated to the growth of perianth and is up-regulated in PeMADS9 (OAGL6-2 ortholog) VIGSenlarged lips
To investigate the function of orchid AAF, an OnAAF ortholog PaAAF which showed highest sequence identity/similarity to OnAAF, was identified through the analysis of NGS data and characterized in Phalaenopsis Sogo Yukidian "V3" (Figure 1g). The PaAAF protein encodes 271 amino acids and shows 86% identity and 95% similarity to OnAAF (Figures S1 and S2).
Compared with Oncidium orchids, Phalaenopsis orchids have significantly smaller lips relative to their sepals/petals ( Figure 1g).
Not surprisingly, the expression of PaAAF was lower in the lips than in the petals during the same developmental stages ( Figure 1h).
These results suggest that PaAAF expression is also associated with the promotion of perianth organ growth in Phalaenopsis orchids. In petals, the expression of PaAAF was found to be expressed higher  Figure   S4C,D,F). The total cell number was however similar in these two stages of petals ( Figure S4G). This result indicated that cell expansion rather than cell division was correlated with the increasing size of petal from early stage to later stage of flower development. Thus, the high level of the PaAAF expression in early petal development revealed that PaAAF expression is associated with the regulation of

| AtAAF expression is high in flower buds and AtAAF protein is localized at the plasma membrane and can be palmitoylated
To further validate the function of the AAF ortholog, Arabidopsis AtAAF/TET9 (At4g30430) was characterized extensively in this study.
Based on Arabidopsis eFP browser data (Schmid et al., 2005  Palmitoylation of tetraspanins in mammalian cells has been reported previously (Charrin et al., 2002;Hua, Green, Wong, Warsh, & Li, 2001;Israels & McMillan-Ward, 2010;Yang et al., 2002). Similar to OnAAF and PaAAF in orchids, AtAAF is predicted to be a member of the tetraspanin family and contains four palmitoylation sites, C65, C66, C252 and C253 ( Figure S1), predicted by CSS-alm (Ren et al., 2008). It is possible that AtAAF may also require palmitoylation to perform its function and that a mutation in the palmitoylation sites may generate a palmitoylation-deficient mutant for AtAAF. Therefore, we gener- biotin switch assay (Hemsley et al., 2008) was performed using anti-GFP antibody (Figure 2i). The results indicated that the AtAAF protein was indeed palmitoylated, whereas the AtAAF palm protein was not (Figure 2i).

| Ectopic expression of AtAAF increases the size of flower organs and seeds
To were clearly larger and heavier than wild-type seeds (Figure 3n), increasing by approximately 74% (Figure 3p). Furthermore, the altered phenotype for the 35S::AtAAF plants was correlated with high level of AtAAF expression (Figure 3q).

| Ectopic expression of palmitoylation-deficient C65S, C66S, C252S, and C253S multiple point mutations in AtAAF causes early senescence and reduced size of flower organs and seeds
To further determine the role of AtAAF, AtAAF loss-of-function T-
The result indicated that these AAF mutants were phenotypically    Reimann et al., 2017;Zuidscherwoude et al., 2015). The organization of the integrin-tetraspanin microdomain and modulation of adhesion-dependent signaling were mediated by palmitoylation of tetraspanins (Berditchevski et al., 2002). It has been shown that ectopic expression of the human palmitoylation-deficient tetraspanin CD151 in Rat-1 cells impaired the interactions of the endogenous tetraspanins CD63 and CD81 and weakened the association of integrin with the tetraspanin-enriched microdomains and affected integrindependent signaling (Berditchevski et al., 2002). It also showed that the mutation in palmitoylation site reduced the ability of human tetraspanin CD81 to interact with other proteins (Delandre et al., 2009) and tetraspanin CD82 to inhibit cancer cell migration and invasion (Zhou et al., 2004). These results indicated that overexpression of Pronounced small-leaf and early-senescence phenotypes were observed in the severe 35S::AtAAF palm transgenic plants (Figure 4a).
One of the reasons for defects in anther dehiscence is the mutation of JA biosynthesis genes such as DAD1 and OPR3 (Ishiguro, Kawai-Oda, Ueda, Nishida, & Okada, 2001;Sanders et al., 2000). We thus investigated whether an external application of JA rescued the sterility of the 35S::AtAAF plants in a similar manner to that in JAtreated dad1 flowers (Ishiguro et al., 2001). Dehiscence of anthers ( Figure S6G) and elongation of siliques ( Figure S6I) were observed in JA-treated 35S::AtAAF flowers. These phenotypes were clearly distinguished from those observed in the JA-untreated 35S::AtAAF flowers, which did not show anther dehiscence ( Figure S6H) or silique elongation ( Figure S6I). Further analysis indicated that the expressions of genes involved in JA biosynthesis, such as DAD1 and OPR3, were all significantly down-regulated in the flowers of 35S::AtAAF plants ( Figure S6J).

| The secondary wall thickness is deficient and the expression of NST1/2 that participate in lignin accumulation of anther secondary wall is down-regulated in 35S::AtAAF transgenic Arabidopsis
In wild-type plants, secondary thickening occurs in the endothecium before anther dehiscence, and the surrounding cell layers of the anther do not undergo secondary thickening (Cecchetti et al., 2013;Yang et al., 2007). To examine the formation of secondary

| Ectopic expression of AtAAF enhances drought/salt tolerance and auxin response
35S::AtAAF expression also enhanced the drought and salt tolerance of transgenic Arabidopsis. One-week-old wild-type seedlings clearly withered 15-30 min after they were removed from the MS medium and exposed to a stream of air ( Figure 6a) and subsequently lost approximately 20%-40% of their weight (Figure 6c). By contrast, the 35S::AtAAF seedlings did not show signs of wilt 30 min after treatment, and their weight was similar to that of untreated seedlings (Figure 6b,c). The 35S::AtAAF seedlings started to show signs of wilt 60 min after treatment, and they lost approximately 30% of their weight (Figure 6b,c). During this same time, wild-type seedlings severely withered and lost approximately 70% of their weight (Figure 6a,c). Unlike the 35S::AtAAF plants, the 35S::AtAAF palm palmitoylation-deficient mutant seedlings clearly showed lower drought tolerance, withered earlier and lost more weight than wild-type seedlings from 15 to 60 min following drought treatment (Figure 6c).
The wild-type seeds barely germinated into seedlings (Figure 6d,f) and had a survival rate as low as 10% (Figure 6g) in MS medium containing 150 mM NaCl. By contrast, more than 60%-80% of the 35S::AtAAF seeds germinated and produced leaves (Figure 6d,e,g).
Unsurprisingly, a clear up-regulation of AtAAF expression was observed 15 min after drought or salt treatments (Figure 6h,i).
It has been reported that auxin regulates flower organ development, since the elongation of petals and stamens is defective in auxin response factor 6 (arf6) and arf8 double-mutant plants (Tabata et al., 2010). Auxin also regulates anther dehiscence by controlling the timing of endothecium secondary cell wall lignification and JA biosynthesis (Cecchetti et al., 2013), and auxin is involved in the response to abiotic stresses such as drought and salt Shi et al., 2014). Since 35S::AtAAF altered flower organ size, anther dehiscence, F I G U R E 6 The analysis of drought and salt tolerance for 35S::AtAAF plants. (a, b) One-week-old wild-type (a) and 35S::AtAAF (b) seedlings were tested for drought tolerance by removing them from MS medium and exposing them to a stream of air for 15, 30, 45, 60, 75, or 90 min. Bar = 5 mm. (c) The 35S::AtAAF (AtAAF) seedlings clearly withered later and lost less weight than wild-type (WT) seedlings at 15, 30, and 60 min after drought treatment. In contrast, the 35S::AtAAF palm (palm) seedlings clearly withered earlier and lost more weight than WT seedlings at 15, 30, and 60 min after drought treatment.
(e) (f) and the response to drought and salt stresses, AtAAF may function in auxin regulation. To investigate this assumption, 35S::AtAAF was introduced into DR5::GFP Arabidopsis, which contains a highly active synthetic auxin response element DR5 with a GFP reporter.
Since auxin is a key signal that coordinates lateral root primordia outgrowth (Benková et al., 2003;Péret et al., 2012;), lateral root production was measured in 2-weekold wild-type and 35S::AtAAF seedlings. The results indicated that 35S::AtAAF plants produced significantly more lateral roots than did wild-type plants ( Figure 7c). Furthermore, IAA-treated 35S::AtAAF plants produced three times more lateral roots than did IAA-treated wild-type plants (Figure 7c). This result supports an enhanced auxin response in 35S::AtAAF that could stimulate the formation of lateral roots. Formation of lateral roots was significantly prohibited in 35S::AtAAF and wild-type seedlings grown at medium containing 2-NOA (auxin influx inhibitor) as controls ( Figure 7c).

| Lateral root primordia of 35S::AtAAF develops faster than wild type
It has been reported that a 90-degree gravitropic stimulus can induce lateral root primordia (LRP) to form on the outer side of bending roots (Lucas, Godin, Jay-Allemand, & Laplaze, 2008;Péret et al., 2009). In addition, auxin plays a key role as a signal that coordinates lateral root primordia outgrowth, outer tissue deformation, and cell separation (Benková et al., 2003;Lucas et al., 2008;). There are eight LRP stages previously described (Péret et al., 2012). When grown on a 90-degree gravitropic stimulus, wild-type seedlings produced stage I (Figure 7d-1) lateral root primordia 18 hr post-gravitropic induction (pgi), while 35S::AtAAF seedlings produced stage VII LRP (Figure 7d-2) in the same time frame. Following auxin treatment, the wild-type seedlings developed stage VI LRP (Figure 7d-3), while the LRP produced by 35S::AtAAF seedlings were beyond stage VIII (Figure 7d-4) at 18 hr pgi. These results again suggest that 35S::AtAAF could enhance the auxin response and hasten the formation of LRP on the roots.

| D ISCUSS I ON
In this study, orthologs of the tetraspanin gene AAF, which are associated with the regulation of perianth size, were identified and characterized in Oncidium and Phalaenopsis orchids. The results obtained indicate that the higher the expression level of orchid AAF orthologs, the larger size of the perianth organ produced. In Oncidium orchids, after perianth identity was determined by P-code complexes, OnAAF was expressed at a higher level in lips than in sepals/petals and was associated with the production of larger lips. By contrast, PaAAF was more highly expressed in sepals/petals than in lips and was associ- Oncidium orchid whereas high PaAAF expression in petals is associated with the production of large size of petals in Phalaenopsis orchid after their organ identity was determined by P-code complexes.
One interesting and critical issue that must be explored is the exact role of AAF orthologs in positively regulating perianth size and other plant developmental processes. The answer is largely revealed by the analysis of Arabidopsis AAF ortholog AtAAF. It is interestingly to note that AtAAF has been reported to be possibly regulated by MADS box gene AGL15 in Arabidopsis (Wang et al., 2015). Two putative MADS protein binding site of CArG boxes consensus sequence (CC(A/T) 6 GG) were identified in the promoter region of AtAAF ( Figure S8). These results indicated that AAF or- No or low effect on cell expansion was observed during late flower development since AtAAF is normally expressed low during late developmental stage (Figure 8). This caused the production of small petal with fewer cells of normal size in AtAAF palm palmitoylation-deficient mutant when compared to those in wild-type petals.
The next question we propose is in what mechanisms AtAAF participates in regulating JA activity. It is worth noting that auxin could inhibit the expression of JA biosynthesis genes DAD1 and OPR3 to result in anther indehiscence (Cecchetti et al., 2013). In addition, petal size was increased in opr3 mutants (Brioudes et al., 2009) and was significantly reduced in arf6/arf8 double-mutant plants (Tabata et al., 2010). These results suggest a possibly functional correlation between AtAAF and auxin in regulating JA activity. Interestingly, auxin-controlled phenotypes, such as the enhancement of drought and salt tolerance, the development of fast growing lateral root primordia, and the production of significantly more lateral roots, were also observed in 35S::AtAAF plants. Thus, we propose that AtAAF is likely participating in auxin regulation by either increasing auxin production or enhancing the efficiency of the auxin response. It is clear that a similar amount of IAA was detected in wild-type, 35S::AtAAF, and 35S::AtAAF palm plants, indicating that AtAAF is likely functioning to enhance the efficiency of the auxin response rather than to increase auxin production. This conclusion is supported by three lines of evidence. First, exogenous auxin treatment increased the amount of GFP transcript much higher in DR5::GFP/35S::AtAAF than in DR5::GFP plants. Second, 35S::AtAAF plants produced three times more lateral roots than did wild-type plants, and the formation of LRP on the roots was much faster in 35S::AtAAF than in wild-type seedlings after auxin treatment. Third, after auxin treatment, unlike the asymmetric gravitropism impairment in DR5::GFP roots, asymmetric gravitropism was still retained in DR5::GFP/35S::AtAAF roots.
All these results indicate that AtAAF could function to increase efficiency of the auxin response, directly or indirectly, in controlling JA activity and plant developmental processes.
As illustrated in Figure 8, our results reveal a possible model for the interaction of the tetraspanin AAF orthologues and auxin in regulating plant growth and development. In Arabidopsis, the targeting of palmitoylated AtAAF proteins to the plasma membrane promotes auxin uptake and enhances the auxin response, which may affect signaling by other plant hormones (JA, ethylene) and control several developmental processes, such as size of flower organs and seeds, anther dehiscence, lateral root formation, and root tip gravitropism, drought and salt tolerance, and organ senescence. Ectopic expression of AtAAF enhanced the auxin response, whereas a palmitoylationdeficient mutation of AtAAF suppressed the auxin response and the processes described above. In Oncidium orchids, after perianth identity was determined by P-code complexes, OnAAF was expressed at a higher level in lips than in sepals/petals, enhancing auxin response and subsequently promoting cell division/expansion in lips more than in sepals/petals. This was associated with the production of larger lips.
By contrast, PaAAF was more highly expressed in sepals/petals than in lips, which was associated with the production of relatively small lips in Phalaenopsis orchids. To further unravel the molecular role for AAF orthologues in auxin response in the future, the analysis of proteinprotein interaction between AAF and the transporters of auxin such as PIN proteins or AUX proteins is necessary. The analysis of signal transduction of cells via AAF from the plasma membrane to nucleus is also needed.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest associated with the work described in this manuscript.

AUTH O R CO NTR I B UTI O N S
C-H. Y. developed the overall strategy, designed experiments, and coordinated the project. W-H. C. performed orchid genes cloning, expression analyses, VIGS experiments, and all the Arabidopsis F I G U R E 8 Model for the function of AAF orthologues in regulating auxin response and development in plants. In plants, the targeting of palmitoylated AAF proteins to the plasma membrane promotes auxin uptake and enhances (→) the auxin response. In flowers, AAF is more highly expressed in early than in late developmental stages (gray bar). Thus, it mainly enhances the auxin response and promotes (green arrow) cell division/expansion in the flower organs at an early stage. Ectopic expression of AAF extends its affect to the whole flower and results in an increase in the size of the flower organs. By contrast, a palmitoylation-deficient mutant of AAF reduces ([ ]) the auxin response and alters its ability to promote cell division/expansion in early flowering stages resulting in a decrease in flower organ size. In addition, the enhancement of the auxin response by ectopic expression of AAF could suppress anther dehiscence during whole flower development by suppressing the expression of MYB26/NST1/2, DAD1/OPR3, and JA activity (which also causes the suppression of the BPEp and resulted in the expansion of the flower organs). The enhancement of the auxin response by AAF also suppresses ethylene signaling and organ senescence, promotes drought/salt tolerance and lateral root formation, and retains root tip gravitropism in plants. Dominant negative mutation of AAF suppresses auxin response and causes the opposite effect on the processes described above