Different physiological processes

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Brassinosteroids (BRs), poly-hydroxylated plant steroidal hormone, play many important roles in plant physiology (Kwon and Choe, 2005). Brassinosteroid signaling consist Kinases - BRI1, BAK1, BIN2, BSKs and transcriptional factors - BZR1/BZR2 and phosphatase - BSU1. In BR signaling pathway, BRs are perceived by BRI1 cell surface receptor and signal is transduced downstream through BSK (Lee and Chory, 1997, Wang et al., 2001; Tang et al., 2008). Recently, Kim et al. (2009) reported that in the presence of BRs, BSK binds to the BSU1 and inactivates GSK3 (BIN2) by dephosphorylating conserved phospho tyrosine residue and resulting in the activation of transcriptional factors BZR1 and BZR2. BZR1 and BZR2 bind with BR biosynthetic genes CPD and DWF4 and inhibit their expression forming a negative feedback loop. In the absence of BRs, BIN2 phosphorylates transcriptional factors BZR1 and BZR2 to inhibit their activities through different mechanisms (proteosome mediated degradation, nuclear export by cytoplasmic retention by 14-3-3 protein and inhibiting DNA binding) to express the BR responsive genes (He et al., 2002; Yin et al., 2002; Vert and Chory, 2006; Bai et al., 2007; Gampola et al., 2007; Ryu et al., 2007). However, CPD and DWF4 genes set free from the repression of BZR1/BZR2 and express (He et al., 2005; Tanaka et al, 2005). The homeostasis of bioactive brassinosteroid is controlled by feedback expression of multiple genes (DET2, DWF4, CPD etc.) and is under the control of BRI1 mediated signaling pathway (Tanaka et al., 2005). The DWF4 expression is critical mechanism in maintaining homeostasis of brassinosteroid (Kim et al., 2006). So the CPD and DWF4 expression level is used as molecular marker to understand the BR signaling (Wang and Chory, 2006).

The plant development is controlled by the interaction of many hormones. BR synergistically interacts with auxin in lateral root development (Bao et al. 2004), and hypocotyls elongation (Nemhauser et al. 2004, Stavang et al, 2009). Combined treatment of BR and auxin synergistically interact and increase hypocotyls elongation through BIN2 regulated phosphorylation of ARF2 (Vert, 2008). Moreover, we found that auxin induces BR biosynthetic DWF4 gene which is more prominent in high temperature even in bri1-5, a BR signaling mutant, but lacks hypocotyls elongation response at higher temperature (Chung et al, 2009). Recently, it has been reported that auxin polar transport coupled to brassinosteroid signaling is required to determine the radial pattern of vascular bundle (Ibaries et al., 2009). The study using ABA mutant, aba1 showed induction of DWF4 and CPD by exogenous ABA application (Zang et al, 2009). BRs counteract ABA in regulating plant germination, hypocotyl, root elongation, and stomata apertures. BR-insensitive Arabidopsis mutants det2, bri1-5 and bri1-9 are more sensitive to ABA than the wild type (Xue et al, 2009). Studies have indicated the interaction of BRs and jasmonic acid in plant development. Ren et al.,( 2009) reported the rescue of JA induced root growth inhibition by BL application. Campos et al. (2009) reported antagonistic interaction of brassinosteroids (BRs) and jasmonates (JAs) in trichome density and allelochemical content in tomato.

GSK3 is a multifunctional kinase that performs an important role in several signaling pathways including Wnt, Hedgenog, insulin signaling, mitosis, apoptosis, stem cell renewal and differentiation and circadian rhythm in mammals. In mammals, GSK3 is found in 3 isoforms: GSK3a, GSK3ß, and GSK3ß2 (Meijer et al., 2004). In contrast, plants posses 10 GSK3s known as ASKs for Arabidopsis SHAGGY-like kinases, divided into 4 subgroups (Jonak and Hirt, 2002). Plant GSK3s play important roles in plant developmental processes like flower development, hormone signaling and stress and wounding responses (Jonak and Hirt, 2002). DWARF12/AtSK-1/BIN2/UCU1, a well studied plant GSK3, functions as an important negative regulator in brassinosteroid signaling pathway (Choe et al., 2002; Li and Nam, 2002; Perez and Perez, 2002). The bin2/dwf12-1D, a gain of function mutation in BIN2 displays the phenotype similar to auxin mutants like dwarf with abaxial curling leaves (Choe et al, 2002). The bin2/dwf12-1D mutation displays many peculiar responses. We have found intensified DWF4pro:GUS expression in light and dark grown bin2/dwf12-1D (Kim et al., 2006). The bin2/dwf12-1D shows DWF4pro:GUS expression throughout the length of the root tissue and increases number of root hairs upon exogenous auxin application (Chung et al.20..). The bin2/dwf12-1D is also hypersensitive to ABA in root development (Choe et al, 2002)

The plant GSK3 is not well studied except its role in brassinosteroid signaling even though, the functions of GSK3 has been extensively studied in mammals. Here, we report the study the role of BIN2 in integrating BRs with other signaling pathways by observing the different physiological processes regulated by integration of brasinosteroid and other hormones using GUS reporter gene system of DWF4 gene in bin2/dwf12-1D background. We found that auxin induced DWF4 expression, auxin and BR induced lateral root development and ABA induced DWF4 suppression are regulated through BIN2.


LiCl suppressed the expression of DWF4pro:GUS in Arabidopsis root

Previously, we have shown that DWF4pro:GUS is expressed in actively growing tissues of Arabidopsis and this expression patterns represent the site of BR biosynthesis in Arabidopsis (Kim et al., 2006). We have found intensified DWF4pro:GUS expression in light and dark grown bin2/dwf12-1D and bri1-5.However, upon BL treatment, the GUS activity was not significantly decreased due to the lack of the feedback down regulation of DWF4 (Kim et al., 2006). To investigate the role of BIN2 in DWF4 regulation, we used LiCl, a well known GSK3 kinase inhibitor (Peng et al, 2008), to block the kinase activity of BIN2 in BR signaling. We treated the five days-old- seedlings of DWF4pro:GUS with KCl and LiCl for 24 hours and subjected for GUS staining. The figure 1 illustrates that the DWF4pro:GUS signal was reduced in the primary root of LiCl-treated seedlings compared to the KCl-treated seedling and control as represented by blue staining of GUS activity. As the LiCl inhibited the kinase activity of the BIN2, DWF4pro:GUS expression was reduced. It indicates that DWF4 is positively regulated by BIN2. We have shown that the auxin induces DWF4pro:GUS (Chung et al., 2009). To investigate whether LiCl does affect auxin induced DWF4pro:GUS expression, we further treated the seedling with 2,4-dichlorophenoxyacetic acid ( 2,4-D) alone, or with LiCl. We used KCl as a control. As shown in the figure1, LiCl treatment significantly reduced the DWF4pro:GUS signal while the KCl treatment did not affect in DWF4pro:GUS signal compared to 2,4-D treated primary root of seedling. These results suggest that auxin induced DWF4pro:GUS expression is through BIN2.

To further assess the suppression of DWF4pro:GUS expression by LiCl in BR signaling cascade, we examined whether LiCl alters DWF4pro:GUS expression in brassinosteroid insensitive mutants bri1-5 and bin2/dwf12-1D. The GUS staining pattern of LiCl or KCl alone or combined with auxin treated primary root of BR insensitive mutants bri1-5 and bin2/dwf12-1D was similar with wild type (Figure1). However DWF4pro:GUS expression was still high in bin2/dwf12-1D and bri1-5 background (Figure1).These results imply that the suppression of DWF4pro:GUS expression by LiCl does not depend on BR perception. To confirm DWF4pro:GUS staining pattern reflects DWF4pro:GUS activity, enzymatic assay of GUS expression was carried out. The GUS assay result was consistent with histo-chemical analysis. Quantitative assay showed LiCl decreased relative GUS activity when seedlings were treated with LiCl alone or with auxin. (Figure2). However the relative GUS activity was still higher in bin2/dwf12-1D and bri1-5 which is consistent with the stronger GUS staining in that genetic background in mock condition too.

Abscisic Acid (ABA) suppressed DWF4pro:GUS expression

It has been reported that ABA increased the DWF4 expression in ABA deficient mutant aba1 (Zhang et al, 2009). We wanted to see whether the ABA induced DWF4 expression is observed in GUS histochemical analysis in wild type background. We treated the five days-old seedlings of DWF4pro:GUS with ABA, for 24 hours and subjected for GUS staining. In contrast with previous result in aba1 (Zhang et al, 2009), Figure 3 illustrates the reduced expression of DWF4pro:GUS in the primary root of ABA treated seedling compared to the KCl treated seedling. To investigate whether ABA induced DWF4pro:GUS suppression is affected by LiCl, we further carried out the combined treatment with ABA and LiCl. As shown in the figure 3, LiCl treatment further reduced DWF4pro:GUS signal while the KCl did not show any effect in DWF4pro:GUS signal compared to ABA treated primary root of seedling. While the same experiment was done in BR insensitive mutant bin2/dwf12-1D/ and bri1-5 background, both of these mutants were slightly sensitive to ABA in term of DWF4pro:GUS expression as revealed by stronger blue staining as seen in figure 3 and higher relative GUS activity in Quantitative GUS assay (Figure 4). All these results suggest that 1 mM ABA strongly suppressed DWF4pro:GUS than 10 mM LiCl in all the mutants background.

Brassinosteroid induced lateral root development

The exogenous application of low concentration of brassinosteroid promotes root length however the higher concentration inhibits. ((Mussig et al., 2003; Roddick and Guan, 1991; Sasse, 1994). Bao et al.( 2003) reported the induction of lateral root formation by brassinosteroid application. To further assess the mechanism of BL induced lateral root formation, we performed the BL dose response test to primary root length and lateral root growth in DWF4pro:GUS lines in bin2/dwf12-1D and bri1-5 genetic background. As it was reported previously (Bao et al., 2003) , 1nM to 100 nM of BL inhibited the elongation of primary root in wild type however, bin2/dwf12-1D and bri1-5,BRinsensitive mutants responded differently with BL. The 1 nM and 2.5 nM of BL inhibited the root growth of bin2/dwf12-1D, in contrast, 5 nM and 10 nM BL enhanced the root growth. In our experimental condition 50 nM and 100 nM BL did not produce any effect in root length in bin2/dwf12-1D.In the bri1-5 background,1 nM and 2.5 nM BL showed promotive effect and 5 nM,10 nM 50 nM and 100 nM showed no significant effects in the primary root growth.

Among 2 BR insensitive mutants, dwf12-1D develops higher number of lateral root / cm of primary root length in contrast bri1-5 developed reduced number of lateral root compared to wild type. In this study,the low concentration of BL 1 nM to 50 nM promoted the lateral root number /cm of primary root of DWF4pro:GUS. The BL induced promotion of lateral root number was highest at 5 nM concentration (Figure 5 A and B). In bin2/dwf12-1 , 2.5 nM BL produced highest number of lateral root /cm primary root. The increased lateral root numbers is not the effect of decrease in root length. For example, at 2.5 nM BL, root length is decreased by 40 % and 45 % respectively in DWF4pro:GUS and bin2/dwf12-1D, however the lateral root number /cm of primary root length is increased by 155 % and 76% respectively in DWF4pro:GUS and bin2/dwf12-1D. The exogenous application of BL could not rescue the reduced number of lateral root in bri1-5. Interestingly, BL decreased the lateral root number in bri1-5 (Figure 5 A and B).The higher concentration of BL is inhibitory in lateral root development in all the cases. All these results strongly suggest - i) dwf12 and bri1-5 are sensitive to BL at low concentration, ii) BL may respond to primary root growth and lateral root development through different mechanisms, iii) BL induced lateral root development needs BR perception.

Auxin interact with BL in lateral root development through BIN2

Auxin is an important phytohormone to promote the lateral root development. The auxin mutants insensitive or less sensitive to auxin develop less or no lateral root (Fukaki,2007). The BR insensitive mutant bri1-5 also exhibits the reduced number of lateral root. Since BL and auxin synergistically promote the lateral root development, we were interested to see the possible point of interaction in BR signaling. As shown in figure 6 A and B, the lateral root number was dramatically higher in DWF4pro:GUS in bin2/dwf12-1D/bin2-3 background while it was lower in bri1-5 background compared to wild type in mock. The application of 2,4-D induced lateral root development and the expression of DWF4pro:GUS in lateral root in all mutants as well as wild type (Figure 6A and 7). To study the role of BIN2 in auxin induced lateral root development, we administered LiCl in the experiment. The figure 6A revealed the great reduction in lateral root number after combined treatment of LiCl and auxin compared to auxin alone but no change in auxin induced lateral root after KCl application. When the BIN2 activity was inhibited by LiCl the auxin induced lateral root growth was greatly reduced explaining the positive role of BIN2.However, the negative effect of LiCl on lateral root density as well as DWF4pro:GUS expression was less pronounced in DWF4pro:GUS in bin2/dwf12-1D (figure 6 A and B and 7).

These results imply that BIN2 has positive role in lateral root development, the synergistic effect of auxin and BL in lateral root development does not rely on BRI1 receptor. BR and auxin interaction in lateral root development is downstream to the BR perception and probably through BIN2.

ABA inhibits lateral root development

According to De Smet (2003) exogenous ABA inhibits LR development immediately which is reversible. It means BL and ABA have antagonistic effect in lateral root development. To test if mutation in BR signaling alters the ABA inhibition of lateral root development, we counted the lateral root number / cm primary root length in ABA-treated dwf12-1D and bri1-5.Figure 8A illustrates the inhibition of lateral root by ABA in wild type as well as in BR mutants, bin2/dwf12-1D and bri1-5. 1mM ABA decreased lateral root number/cm primary root by 64%, 39% and 26% of mock in DWF4pro:GUS, dwf12-1D, and bri1-5 respectively. However, ABA did not show significant effect on primary root elongation. Importantly, bin2/dwf12-1D and bri1-5 were less sensitive to ABA in lateral root inhibition which suggests that the ABA mediated lateral root inhibition requires the functional BRI1 and BIN2.


Materials and Methods

Plant material and growth condition

Previously, we have reported DWF4pro:GUS transgenic line in Ws-2, bin2/dwf12-1D and bri1-5 background(Kim et al. 2006). Plant growth condition are as described in Choe et al.(2001). Briefly, seeds were sterilized with 70 % ethanol for 2 min, 5% commercial bleach and 2 % SDS solution for 10 min and washed 10 times with sterile water. Seeds were plated on 1X Murashige and Skoog (MS) supplemented with 1 % (w/v) sugar and 0.4% (w/v) phytoagar. The seedlings were grown in 22OC under long day condition (16 -h light / 8 -h dark for 5 days and then transferred to the MS media supplemented with different chemicals or hormones and grown for indicated time.

Primary root growth and lateral root counting

For counting lateral root in BL-treated seedlings, sterilized seeds were sown in 1X MS containing different concentration of epi-BL and were grown in vertically placed agar plates for 8 days after 3 days inhibition in cold room. For auxin and ABA treatment, five days-old seedlings were transferred to the 1X MS containing 10 mM KCl, 10 mL LiCl . 0.1 uM 2,4-D , 1 uM ABA and grown for 24 hours before GUS staining and in vivo assay.The root length was measured by using image tool software (Uthscsa image tool version 3.0) after photographed. For the lateral root counting, the above seedlings were subjected to histochemical staining and lateral root primordia and lateral roots were counted under optical microscope.

Histochemical and quantitative GUS assay.

GUS staining was performed as described by Jefferson (1987) with minor modification.

Seedlings were incubated at 37OC for 16 hours in GUS staining buffer (1mM 5 bromo-4-chloro-3 indoylß-DGlcUA, 100 mM Sodium Phospahate (PH7.00), 0.5 mM Potassium ferrocyanide ans 0.5 mM Potassium ferric cyanide, 10 mM EDTA and 0.1 % (v/v) Tritron X -100). The chlorophyll in tissue was cleared by 50%, 70% and 100% ethanol and observed lateral root and photographed by optical microscope.

Quantitative GUS assay was performed according to Blazquize et al. (1998). Whole seedling was transferred to a well of 96- well plate containing 100 ul of a substrate solution (50 mM Sodium Phosphate (PH 7.00), 10 mM ß- Mercaptoethanol, 10 mM EDTA, 0.1% (w/v) SDS, 0.1 % (w/v) triton X-100, 2% isopropanol and 440 mg /l 4- methylumbeliferyl ß-D-glucuronide incubated at 37 OC for 12 hours. The reaction was stopped by adding 100 ul of ice cold stop buffer (0.2 mM Na­2CO3). Florescent products were quantified with flurometer (Cary Eclipse Fluorescence Spectrophotometer,Varian Inc., USA) with setting of excitation wavelength at 360 nm and emission wavelength at 465 nm. The standard curve was calculated using the known quantity of 4- Methylumbelliferol solution.


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