The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively regulate lateral root development by repressing a vacuolar invertase in Arabidopsis

Lateral root (LR) architecture determines the efficiency of nutrient absorption and anchors the plant. Internal auxin regulatory mechanisms that control the development of LR have been identiﬁed, but how external nutrients influence lateral root development remains elusive. We have characterized the functions of the FK506-binding proteins FKBP15-1 and FKBP15-2 in Arabidopsis. FKBP genes are mainly expressed in the vascular bundle of the root basal meristem region, and the FKBP proteins are localized to the endoplasmic reticulum. Co-IP and BIFC assays showed that FKBP15-1 and FKBP15-2 interact with the vacuolar invertase 2 (VIN2). Compared to Col-0 and the single mutants, the double mutant fkbp15-1fkbp15-2 had more LRs and LR initiation density, and possessed higher sucrose catalytic activity. Moreover, VIN2 can complement the phenotype of increased LRs in the fkbp15-1fkbp15-2 double mutant. Our results indicate that FKBP15-1 and FKBP15-2 together participate in the control of LR numbers by regulating the enzyme activity of VIN2. Due to the activity of peptidylprolyl cis-trans isomerases owned by FKBP family proteins, our results provide a clue to further analysis the interplay between lateral root development and protein modification. Peptides were a peptide column (Thermo Fisher Scientific, USA), and eluted using a solvent system consisting of solvent A (99.9% water, 0.1% formic acid), and solvent B (99.9% acetonitrile, 0.1% formic acid). The peptides were eluted with a gradient of 2-30% solvent B for 80 min, 30-80% solvent B for 10 min, and finally 80% solvent B for 10 min with a constant flow rate of 250 nl/min in a C18 capillary column (Thermo fisher Scientific, USA). The eluted ions were analysed on an ESI-Q-TOF mass spectrometer in data dependent acquisition mode (m/z 350-1500). The Source Capillary was set at 2000-2400 v, the flow rate and temperature of the dry gas were 2.0 L/min and 150°C, respectively. The mass spectrometer was set as one full MS scan followed by ten MS/MS scans on the ten most intense ions from the MS spectrum with the dynamic exclusion duration set at 15 s. Tandem mass spectra were extracted, and the charge state was de-convoluted and de-isotoped using Compass Data Analysis version 4.1 (Bruker Daltonics). The peak list was directly generated from the raw data using a centroid algorithm with peak width set at 0.1 m/z and intensity above 100. No peak smoothing or filter processing was applied. After the charge states were calculated, the de-isotoped peak lists were exported as mgf files for further Mascot searches. Mascot (version 2.4, Matrix Science) was set up to search the database. The following parameters were considered for the searches:

2 Abstract Background Lateral root (LR) architecture determines the efficiency of nutrient absorption and anchors the plant.
Internal auxin regulatory mechanisms that control the development of LR have been identified, but how external nutrients influence lateral root development remains elusive.

Results
We have characterized the functions of the FK506-binding proteins FKBP15-1 and FKBP15-2 in Arabidopsis. FKBP genes are mainly expressed in the vascular bundle of the root basal meristem region, and the FKBP proteins are localized to the endoplasmic reticulum. Co-IP and BIFC assays showed that FKBP15-1 and FKBP15-2 interact with the vacuolar invertase 2 (VIN2). Compared to Col-0 and the single mutants, the double mutant fkbp15-1fkbp15-2 had more LRs and LR initiation density, and possessed higher sucrose catalytic activity. Moreover, VIN2 can complement the phenotype of increased LRs in the fkbp15-1fkbp15-2 double mutant.

Conclusion
Our results indicate that FKBP15-1 and FKBP15-2 together participate in the control of LR numbers by regulating the enzyme activity of VIN2. Due to the activity of peptidylprolyl cis-trans isomerases owned by FKBP family proteins, our results provide a clue to further analysis the interplay between lateral root development and protein modification.

Background
Lateral roots (LRs) are one part of plant root system that function in nutrient and water uptake as well as in physical anchorage [1]. To meet the demands of plant growth and survival, the root system needs to develop a large number of lateral roots to increase root space in the soil [2]. Plants with well-developed lateral root system usually have better nutrient absorption capabilities and higher yields [3].
The development of lateral roots can be divided into at least three distinct phases; lateral roots initiation, the formation of lateral root primordia, and post-emergence growth [4]. Lateral roots initiate from the pericycle cell layer of main root basal meristem area [5]. Some xylem pole-pericycle (XPP) cells in the initiation zone accumulate the maximum amount of auxin and undergo the selection of cell differentiation [6,7]. Auxin drives the transcriptions of auxin response factor (ARF) genes to control the expressions of GATA transcription factors during establishing the lateral root founder cells [8]. After the first asymmetric division, the divided daughter cells generate a set of short cells called stage I primordium. Cells in stage I divide periclinally to form a two-cell layered primordium. The following developmental stages then give rise to a dome-shaped primordium, and a new lateral root finally emerges from primary root. During these successive developmental processes, auxin induces the specification of the LR primordium through activating ARF7 and ARF19 expressions [9,10]. Both ARF7 and ARF19 can be phosphorylated by the brassinosteroid (BR) signal transducer BIN2, and inhibited by ABA [11,12]. Besides them, cytokinin acts as a negative regulator by regulating auxin synthesis, transport, and signalling to influence LR formation and growth [13]. These results together indicate that the emergence of LRs is a complicated process, which needs the cooperation among different phytohormones.
Lateral root formation is regulated not only by phytohormones but also by environmental factors [14].
CYTOKININ RESPONSE FACTOR2(CRF) CRF2/CRF3 function in the LRs for the adaptation to cold stress, in which oxidative-stress-induced reactive oxygen species (ROS) facilitate LR outgrowth by promoting cell wall remodelling in adjacent tissues [15]. When grown under low-phosphorus conditions, LR numbers increase, and their growth angles from main root become smaller in order to acquire more phosphate from the surrounding soil [16]. LR formation is also regulated by nitrate; nitrate-rich soils repress LR formation, whereas locally sparse nitrate levels promote the emergence of LRs [17]. In addition to nitrogen and phosphate, sugars affect LR formation by changing auxin biosynthesis or signalling [18,19]. Sugars promote lateral root formation at low concentrations, whereas they become inhibitory factor at higher levels. Increasing the concentration of glucose somewhat increases the number of LRs by regulating the expression of auxin-related genes including biosynthesis, transporter, and receptor genes [20,21], indicating the importance of sugar transporting and 4 metabolism in the development of LRs.
Immunophilins are two evolutionarily unrelated groups of proteins with high affinities for the immunesuppressive drugs FK506 and rapamycin, or cyclosporine A (designated as FKBPs/FK506 binding proteins and cyclophilins, respectively) [22]. Immunophilins with the activity of peptidylprolyl cis-trans isomerase widely distribute across different species [23,24]. In plants, immunophilins comprise large protein families, and play essential roles in stress response, redox regulation of chloroplast function as well as LR development [25][26][27]. In tomato, SlCyp1 regulates the activities of PIN transporters by affecting their localization in plasma membrane during LR formation [28]. In rice, the cyclophilin protein LRT2 directly regulates the stability of the OsIAA11 protein by changing its cis-trans structure; the lrt2 mutation accumulates a higher level of OsIAA11, which then suppresses auxin-induced lateral root development [29]. FKBP42/TWD1 in Arabidopsis positively modulates ABCB/P-glycoprotein transporter activity in polar auxin transport. The FKBP42/TWD1 mutant displays defects in LR development [30][31][32]. Taken together, these results indicate that immunophilins participate in LR development mostly by regulating gene expressions related to auxin transporting.
To further analysis the roles of immunophilins in LR development, we investigated the function of FKBP15-1 and FKBP15-2 that are phylogenetically related to FKBP42/TWD1 in Arabidopsis. The results showed that both FKBP15-1 and FKBP15-2 could interact with the vacuolar invertase VIN2. Knocked out FKBP15-1 and FKBP15-2 expressions increased the number of LRs and improved the activities of VIN2, suggesting that both FKBP15-1 and FKBP15-2 are required for LR development via the regulation of VIN2 catalysing activity. Because FKBPs possess the activity of peptidyl prolyl cis-trans isomerase, our findings provide clues for further analysing the relationship between LR development and protein folding.

Plant materials
Arabidopsis thaliana ecotype Col-0 and the T-DNA insertion mutants in FKBP15-1 and FKBP15-2 were provided by the European Arabidopsis Stock Centre [33]. Due to the limited number of FKBP15-2 mutant, RNA interference (RNAi) plants of FKBP15-2 were also generated. The FKBP15-2 specific coding region was amplified using primers carrying attB sites (Additional file 6: Table S1) and recombined into the pHellsgate12 vector to generate the RNAi construct [34]. The construct was To generate the triple mutant, a plant homozygous for the vin2 gene mutation (SALK_100813) was crossed with the fkbp15-1fkbp15-2 double mutant. After self-pollination of the F 1 , triple mutant plants (fkbp15-1fkbp15-2vin2) were generated and confirmed by PCR.

RNA extraction and quantitative real-time PCR (qRT-PCR) assays
The tissues (root, stem, leaf, and flower) sampled from 40-day-old seedlings of the Col-0 wild-type and the different mutants and RNAi lines were ground to powder in liquid nitrogen. Total RNA was isolated using the RNA prep-pure Plant Kit as described by the manufacturer (Tiangen, China). RNA samples were quantified by absorbance at 260 nm using the Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), and the relative purity was assessed by the A 260/280 ratio. For cDNA synthesis, 1 μg samples of total RNA were treated with DNase I to remove contaminating genomic DNA, and then reverse-transcribed into 1 st -strand cDNA using the PrimeScript RT reagent Kit as directed by the manufacturer (Takara, Japan).

6
The qRT-PCR assays were performed on a LightCycler 96 system (Roche, Switzerland) using the SYBR Premix Ex-Taq kit (Takara, Japan). The amplification conditions were: an initial denaturation at 95°C for 10 min, followed by 15-25 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 30 sec, after which samples were returned to room temperature. mRNA of the housekeeping gene UBQ10 was used as internal control. Relative transcription levels were calculated using the comparative 2 -ΔΔCT method [35]. All experiments were repeated at least three times for reproducibility. The DNA sequences of all primers are given in Additional file 6: Table S1.
The constructs were transformed into Col-0 plants, and the transgenic plants were verified by PCR.
To determine the expression patterns of FKBP15-1 and FKBP15-2 in roots in detail, GUS-stained roots were fixed overnight in 1% glutaraldehyde and 4% paraformaldehyde (pH 7.4). The samples were dehydrated in a graded ethanol series and embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany). The embedded samples were cut into 10 mm sections with a microtome (Leica EM UC7, Germany), and prepared for observation and imaging with a differential interference contrast (DIC) microscope (NIKON 80i, Japan).

Subcellular localization of FKBP proteins and BIFC analysis
Subcellular localization assays were performed using a previously-described method [37].
Agrobacterium strain GV3101 separately carrying the 35S::FKBP15-1-eYFP::NOS, 35S::FKBP15-2-eYFP::NOS, and ER marker ER-rk expression constructs were grown at 28°C overnight. The ER-rk was created by first inserting ER retaining signal at the C-terminus of the mCherry and subsequently adding the signal peptide of AtWAK2 at the N-terminus. The liquid cultures were collected by centrifugation at 2,000 g for 10 min, and the pellets were re-suspended in MS medium and adjusted to OD 600 =0.5-0.6. A final concentration of 200 μM acetosyringone and 10 mM MES (pH 5.6) were added, and the bacterial suspensions were kept at room temperature for at least 3 hours without shaking prior to infiltration into N. benthamiana leaves. Three days after infiltration, the fluorescence signals in leaf epidermal cells were visualized using a Leica SP5 confocal microscope.
For BiFC assays, full-length FKBP15-1 and FKBP15-2 were fused with C-terminal YFP in the vector pEarleygate202-YC, while full-length VIN2 was cloned into vector pEarleygate201-YN and both were transformed into Agrobacterium strain GV3101. Equal ratio of re-suspended bacterial of FKBP15-1/15-2-cYFP and VIN2-nYFP were co-injected into N. benthamiana leaves as described above.  hours, pieces of the transformed leaves were observed with confocal microscope for fluorescent signal.

Root growth measurements
To analyse the root phenotypes in Col-0 and the FKBP mutants, seeds were surface-sterilized for 5 min in 10% (v/v) sodium hypochlorite and washed five times with sterile water, then placed on 0.5× MS agar plates under different treatment conditions. To determine the effects of auxin on root growth, IAA was added to 0.5× MS medium at a concentration 5 nM or 50 nM. The effects of sugars on root growth were determined by adding sucrose, glucose, and fructose separately to 0.5× MS medium at different concentrations (0%, 0.5%, 1%, 2%, 3%, and 5%). Plates were placed vertically in a growth chamber at 22°C at a light/dark cycle of 16/8 h.
After five days of growth, at least 50 plants in each treatment were examined every day. Root lengths 8 were recorded and photographed. All experiments were repeated at least three times. Statistical differences between the different genotypes were calculated using Student's t-test.

Protein co-immunoprecipitation assays
To determine which proteins interact with FKBP15-1 and FKBP15-2, we performed protein coimmunoprecipitation assays with an anti-GFP antibody [38]. Roots from 12-day-old FKBP15-1-eYFP and FKBP15-2-eYFP transgenic seedlings were collected and ground to powder in liquid nitrogen. Total proteins were extracted in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, pH 7.5, and one complete protease inhibitor cocktail tablet per 25 mL). Following gentle shaking at 4°C for 30 min, the extraction solutions were centrifuged at 15,000 ×g for 10 min. The supernatants were kept on ice before they were used in immunoprecipitation assay. The anti-GFP antibody (Genescript, China) and protein G Sepharose (GE Healthcare, USA) were first mixed and incubated at 4°C for 30 min, and the Sepharose-antibody mixture was then incubated with the supernatant with gentle shaking at 4°C for 1 h. The protein G Sepharose was collected by centrifugation (30 sec, 15,000 ×g) and washed three times before elution with a buffer consisting of 50 mM Tris-HCl, pH 6.8, 50 mM dithiothreitol, 1% SDS,1 mM EDTA, 0.005% bromophenol blue, and 10% glycerol. The eluted proteins were separated by SDS-PAGE for either silver nitrate gel staining, western blotting, or mass spectrometry analysis.

Protein digestion and LC-MS/MS analysis
Protein bands excised from the SDS-PAGE gels were destained by incubation in destaining solution (7.5 mM potassium ferricyanide and 25 mM sodium thiosulfate). The proteins in the gel pieces were reduced by incubation in 10 mM DTT solution at 60°C for 20 min, followed by alkylation in a solution of 25 mM IAM at room temperature for 15 min. The gel pieces were treated with trypsin (Promega, Madison, WI) overnight at 37°C to digest the proteins. The resulting peptides were extracted with 60% acetonitrile containing 5% formic acid, dried in a SpeedVac, and were then re-dissolved in 2% acetonitrile containing 0.1% formic acid for LC-MS/MS analysis.
Peptides were concentrated with a peptide trap column (Thermo Fisher Scientific, USA), and eluted using a solvent system consisting of solvent A (99.9% water, 0.1% formic acid), and solvent B (99.9% acetonitrile, 0.1% formic acid). The peptides were eluted with a gradient of 2-30% solvent B for 80 min, 30-80% solvent B for 10 min, and finally 80% solvent B for 10 min with a constant flow rate of 250 nl/min in a C18 capillary column (Thermo fisher Scientific, USA). The eluted ions were analysed on an ESI-Q-TOF mass spectrometer in data dependent acquisition mode (m/z 350-1500). The Source Capillary was set at 2000-2400 v, the flow rate and temperature of the dry gas were 2.0 L/min and 150°C, respectively. The mass spectrometer was set as one full MS scan followed by ten MS/MS scans on the ten most intense ions from the MS spectrum with the dynamic exclusion duration set at 15 s.
Tandem mass spectra were extracted, and the charge state was de-convoluted and de-isotoped using Compass Data Analysis version 4.1 (Bruker Daltonics). The peak list was directly generated from the raw data using a centroid algorithm with peak width set at 0.1 m/z and intensity above 100. No peak smoothing or filter processing was applied. After the charge states were calculated, the de-isotoped peak lists were exported as mgf files for further Mascot searches. Mascot (version 2.4, Matrix Science) was set up to search the database. The following parameters were considered for the searches: peptide mass tolerance was set to 20 ppm, fragment mass tolerance was set to 0.05 Da, and a maximum of two missed trypsin cleavage sites was chosen. Carbamidomethyl (C) was set as fixed modification, and oxidation (M), was set as variable modifications.

Invertase enzyme activity assays
To assay invertase activity, total proteins were extracted from the roots of 12-day-old Arabidopsis plants in extraction buffer (50 mM Tris-acetate, pH 7.5; 10 mM EDTA; 5 mM DTT). The homogenates were directly used for invertase assays following a previously-described method [39], and protein concentrations were determined by the Bradford method. For the invertase assays, 100 mg samples of total proteins were incubated for 30 min with 1% sucrose in 50 mM potassium phosphate, pH 7.0, at 37°C, followed by reacting with DNS reagent (100 ºC for 5 min) and reading the absorbance at 540 nm. Tubes without sucrose were used as controls. The initial and final glucose concentrations in each 10 sample were quantified on a calibration curve made with different sucrose concentrations. All experiments were repeated four times.

Suppressing FKBP15-1 and FKBP15-2 gene expression in Arabidopsis increases the number of lateral roots
Immunophilin FK506-binding proteins in plants influence central and secondary metabolism, cell cycle, and protein folding during vegetative tissue development [23,25,28,30]. To better understand the roles of FK506 binding proteins in Arabidopsis, we chose to study FKBP15-1 and FKBP15-2 because the proteins encoded by these genes have the highest sequence identity (68.4%) at the amino acid level of all the 32 FKBPs, and also because of their co-expression patterns (accessible through Gene Investigator). We firstly characterized the fkbp15-1 mutant (SALK_035550C) and the fkbp15-2 mutant (SALK_113542). The T-DNA insertion in the fkbp15-1 mutant is located 28 bp upstream of the transcription start site, and the fkbp15-2 mutant harbors a T-DNA insertion in the fourth exon (Fig. 1a). Due to the limited number of knock-out mutant lines available, we also generated a FKBP15-2 gene knock-down line, and used qRT-PCR to verify that the expression of FKBP15-2 was specifically reduced in the FKBP15-2RNAi plants (Fig. 1a, b).
To further analysis the functions of FKBP15-1 and FKBP15-2 in LR development, we overexpressed the FKBP15-1 and FKBP15-2 genes in Col-0 and examined their root phenotypes. Contrary to our expectations, the overexpressed plants (overexpressed FKBP15-1, FKBP15-2, or both genes) showed no significant differences in the number of LRs or primary root length compared to the control Col-0 (Additional file 2: Figure S2). This result indicates that increased transcriptional levels of FKBP15-1 and FKBP15-2 in Arabidopsis cannot change the number of LRs and on primary root development.

FKBP15-1 and FKBP15-2 are mainly expressed in the root vascular bundle
To analysis the spatial-temporal expression patterns of FKBP15-1 and FKBP15-2, the samples from transgenic plants expressing ProFKBP15-1::GUS and ProFKBP15-2::GUS construct were subjected to GUS staining. Histochemical staining revealed that GUS signals were present in the vascular bundles of the cotyledons, leaves, and roots of 10-day-old seedlings, consistent with qRT-PCR results showing that both genes are expressed in all three tissues, with stronger expression occurred in roots (Fig. 3a, d, Additional file 3: Figure S3). In the roots, FKBP15-1 and FKBP15-2 are mainly expressed in the vascular bundles of primary roots (Fig. 3g, h), with high levels of expression observed in the basal meristem during LR initiation (Fig. 3k, l), and lower expression levels in the root tip, apical meristem, and the maturation region (Fig. 3b-c, e-h). To visualize the expression patterns of FKBP15-1 and FKBP15-2 in detail, we observed the GUS signals in cross-sections of the basal meristem, and found that the FKBP15-1 and FKBP15-2 signals were mainly present in the pericycle, xylem, and phloem ( Fig. 3 i, j). Overall, these expression profiles showed that FKBP15-1 and FKBP15-2 are mainly expressed in the root vascular bundle, which is probably related to their roles in the initiation of LR primordia.
benthamiana. Two days after infiltration, fluorescent signals were distributed on the cell membrane and endoplasmic reticulum (ER). Co-localization analysis indicated that both FKBP15-1-eYFP and FKBP15-2-eYFP co-localized strongly with the ER marker ER-rk ( Fig. 4; the Pearson correlation coefficients were 0.966 and 0.957, respectively. The fluorescence intensities of FKBP15-1/FKBP15-2 are identical to that of ER marker) [40]. Due to their co-expression patterns, we further tested the possibility of interactions between FKBP15-1 and FKBP15-2. As shown in Additional file 4: Figure S4, FKBP15-1 interacted with FKBP15-2 in vivo, and the interaction was stronger in the ER and weaker in the cytoplasm. These results implied that the regulation of FKBP15-1 and FKBP15-2 proteins may occur in the ER.

Auxin regulation in lateral root number is unaffected by the transcriptional expression change of FKBP15-1 and FKBP15-2
Auxin is the key phytohormone controlling LR initiation and patterning in Arabidopsis [10,12]. To analysis the effects of auxin LR numbers in the double mutant and fkbp15-1fkbp15-2RNAi, we added indole-3-acetic acid (IAA) in the 1/2 MS medium to final concentration-0 nM, 5 nM, and 50 nM respectively. When the concentration of IAA was increased, we didn't observe the narrowed difference of LR number between the double mutant, or fkbp15-1fkbp15-2RNAi, and Col-0 (Fig. 5a, b). The auxin reporter DR5::GFP showed that the GFP signals in the roots of double mutant and Col-0 plants were identical at the same concentration of IAA (Additional file 5: Figure S5a). Moreover, the expressions of FKBP15-1 and FKBP15-2 genes were unchanged in response to auxin treatment (Additional file 5: Figure S5b). Taken together, these data indicate that knocked-out or knocked-down of both FKBP15-1 and FKBP15-2 expressions does not influence the function of auxin in LR development in Arabidopsis.

FKBP15-1 and FKBP15-2 interact with an invertase protein in Arabidopsis
To identify protein(s) that interact with FKBP15-1 and FKBP15-2 in Arabidopsis, we used the coimmunoprecipitation method with the GFP antibody to catch the candidate proteins from the roots of transgenic FKBP15-1-eYFP and 15-2-eYFP plants respectively. The pulled proteins were separated by SDS-PAGE, and confirmed by Western blotting (Fig. 6a, b). IP-MS analysis showed that 113 and 80 proteins were characterized as the candidates interacted with FKBP15-1 and FKBP15-2, respectively.
Gene Ontology (GO) analysis indicated that 41 overlapped proteins belong to the functions of protein localization, establishment of protein localization, and immune system process (P≤0.01), which agreed with protein modify functions possessed by FKBP families (Fig. 6c, Fig. 7a, Additional file 9, Table. S2). KEGG pathway enrichment analysis revealed that the functions of overlapped proteins are related to the pathways of starch and sucrose metabolism, RNA transport, ribosome, and Cyanoamino acid metabolism (P≤0.01, Fig. 7b, http://kobas.cbi.pku.edu.cn/annotate.php) [41]. Based on P-values and the number of proteins appeared in the immune-precipitated proteins, the vacuolar invertase (VIN2) and two glucosidases (BGLU22 and BGLU23, glycosyl hydrolase superfamily) ranked the top three proteins (Additional file 6: Figure S6).
In plants, vacuolar invertase acts as a key modulator of hexose accumulation and cell expansion [42][43][44]. To analyse the interactions between FKBPs and the invertase, we coexpressed the FKBPs with VIN2 in the epidermis cells of tobacco leaf. As shown in Fig 8a-b, the FKBPs could interact with VIN2, and intense fluorescence signals were observed in the ER of epidermis cells. This result implies that the FKBP15-1 and FKBP15-2 proteins together interact with the invertase to regulate sucrose metabolism during LR development.

FKBP15-1 and FKBP15-2 repress lateral root development by negatively regulating the activity of a vacuolar invertase
To analyse the effects of FKBP15-1 and FKBP15-2 expression on the activity of vacuolar invertase, we compared vacuolar invertase activity in Col-0 with double mutant. Compared with Col-0, the vacuolar invertase activity in the roots of fkbp15-1fkbp15-2 plants increased by about 39%, whereas the insignificant difference was observed in leaves (Fig. 8c). This result indicates that knocked-out both FKBP15-1 and FKBP15-2 gene expressions increases the activity of vacuolar invertase mainly in roots.
To identify whether carbon resources influence LR development, we investigated the LR number of fkbp15-1fkbp15-2 and Col-0 plants in the medium with different kinds of sugars. When the plants grew in the 1/2 MS medium added with 1%-5% sucrose, the differences in the number of LRs between the double mutant and Col-0 were significant (Fig. 8d). When sucrose in the medium was replaced by glucose or fructose, we observed no significant differences in the number of LRs (Fig. 8e, f). Given that the sucrose catalysing ability of invertases and the greater demand for glucose in expanding cells, these results indicated that FKBP15-1 and FKBP15-2 cooperatively repress the development of LRs in a sugar-dependent manner.

VIN2 complement the increased lateral root number in the fkbp15-1fkbp15-2 double mutant
To investigate the genetic relationship between FKBP15-1/FKBP15-2 and VIN2, we generated the triple mutant fkbp15-1fkbp15-2vin2, and determined the LR phenotype (Additional file 7: Figure S7). The results showed that the number of LRs in the triple mutant showed no significant difference compared with Col-0, but was less than that in fkbp15-1fkbp15-2 double mutant ( Fig. 9 a, b). No difference in fresh weight and main root length were either observed between fkbp15-1fkbp15-2vin2, and Col-0 ( Fig. 9 c-d). These results indicate that VIN2 can complement the mutation of fkbp15-1fkbp15-2, and that VIN2 activity is required for LR development.

Discussion
Plant immunophilins function in lateral root development mostly by regulating auxin transport and signal transduction [28][29][30][31][32]. Unlike reported functions of immunophilin in LR development, we show here that FKBP15-1 and FKBP15-2 negatively regulate LR number by interacting with a vacuolar invertase VIN2 in Arabidopsis. Analysis of gene spatial expression and protein subcellular localization showed that both FKBP15-1 and FKBP15-2 localize in the ER, an organelle that plays an important role in protein sorting and folding. Given that FKBP family proteins possess peptidylprolyl cis-trans isomerase activity [23][24][25], these results suggest that both FKBP15-1 and FKBP15-2 proteins may modify the cis-trans conformation of VIN2, and then indirectly change its catalysing activity. In vitro and in vivo analysis showed that the catalytic activity of VIN2 was higher in the control, but repressed in the presence of FKBP15-1 and FKBP15-2 proteins. Although the mechanism how FKBPs change the cis-trans conformation of VIN2 needs to be determined, these results together support that FKBP15-1 and FKBP15-2 regulate the number of LRs probably by changing the conformation of VIN2 during LR initiation.
Sugars influence LR development mainly in two ways: (1) Sugars stimulate LR formation by promoting auxin biosynthesis [18,45]. In this study, FKBP15-1 and FKBP15-2 gene expressions were unchanged upon auxin treatment (Fig. S5). Analysis of auxin reporter DR5::GFP further showed that auxin accumulated in LRs and main root were unaffected by the change of FKBP15-1 and FKBP15-2 expressions (Fig. S5). Moreover, improving auxin concentration in 1/2 MS medium didn't narrow the difference in LR number between fkbp15-1fkbp15-2 and Col-0 (Fig. 8). These results together imply that the regulation of FKBP15-1 and FKBP15-2 in LR development is probably independent of the auxin-transducing pathway in Arabidopsis; (2) High concentrations of sugars may influence LR development by activating ROS production or improving osmotic pressure [46,47]. Given that VIN2 is coexpressed with FKBP15-1/15-2 in pericycle founder cells of the basal meristem region, we deduced that repressed VIN2 activity probably decrease sugar supply in the LR primordia, and in turn results in fewer initiation cells in the wild type. In support of this hypothesis, feulgen staining indicated that the number of LR initiation sites were increased in the fkbp15-1fkbp15-2 mutant. When FKBP15-1FKBP15-2 expression were repressed in the double mutant, VIN2 enzyme activity was increased, and more sucrose are likely to be catalysed into glucose (Glc) and fructose (Fru) that promote the emergence of lateral root primordia [48,49]. Interestingly, two β-1,3 glucanases were recently identified that localized in the plasmodesma; they can regulate callose accumulation, cell-to-cell connectivity, and the number of lateral roots [50]. Both of these β-1,3 glucanases were characterized as the candidates interacted with FKBPs; thus future research is needed to confirm whether these two proteins cooperate with VIN2 to regulate LR initiation. Collectively, this research shows that FKBP15-1 and FKBP15-2 have essential functions of regulating LR development in Arabidopsis. Our findings also provide clues into exploiting the interplay between LR development and protein folding.

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Availability of data and materials
The datasets generated and/or analysed during the current study are not publicly available because we are continuing to mine more data from the datasets. However, they are available from the corresponding author upon reasonable request. Relative changes in FKBP15-1 and FKBP15-2 transcript levels determined by qRT-PCR analysis in the fkbp15-1, fkbp15-2, fkbp15-2RNAi, fkbp15-1fkbp15-2, and fkbp15-1fkbp15-2RNAi plants. The UBIQUITIN 10 gene was used as an endogenous control for normalization of gene expression. Error bars represent the SD of the means from three biological replicates. c Representative phenotypes of 12-day-old Col-0, fkbp15-1, fkbp15-2, fkbp15-      Identification and GO (Gene Ontology) annotation of proteins that interact with FKBP15-1 and FKBP15-2 by immunoprecipitation coupled with LC-MS/MS analysis. a, b Samples before 30 and after immunoprecipitation were separated by SDS-PAGE for silver nitrate staining (a) or western blotting analysis using the GFP antibody (b). (c) GO annotation and the enrichment ratios for the proteins pulled down by FKBP15-1-YFP and FKBP15-2-YFP. The y-axis shows the enrichment ratio for the 26 GO terms in the three major GO categories "Biological Function", "Cellular Component", and "Molecular Function", and the x-axis indicates the proteins that interact with FKBP15-1 and FKBP15-2.  Genetic analysis of FKBP15-1, FKBP15-2 and VIN2. a Phenotypes of 12-day-old Col-0, fkbp15-1fkbp15-2, and fkbp15-1fkbp15-2vin2 seedlings grown on 0.5× MS medium. Scale bar: 1 cm. b Analysis of lateral root numbers in Col-0, fkbp15-1fkbp15-2, and fkbp15-1fkbp15-2vin2 seedlings. Bars represent the average values ± SD of ≥30 seedlings of each genotype. Asterisks indicate significant differences in lateral root number in each genotype