The KNOTTED1 homeobox (KNOX) family transcription factors are essential for stem cell establishment and maintenance and regulate various aspects of development in all green plants. Expression patterns of the KNOX genes in the roots of plants have been reported, but their role in development remains unclear. Here we show how the KNAT1 gene is specifically involved in root skewing in Arabidopsis. The roots of two mutant alleles of KNAT1 (bp-1 and bp-5) exhibited exaggerated skewing to the right of gravity when grown on both vertical and tilted agar medium surfaces. This skewing phenotype was enhanced by treatments with low concentrations of propyzamide, and required auxin transport. The KNAT1 mutation substantially decreased basipetal auxin transport and increased auxin accumulation in the roots. Using a PIN2–GFP reporter and western blot analysis, we found that this alteration in auxin transport was accompanied by a decrease in PIN2 levels in the root tip. Decreased PIN2 expression in the mutant roots was not accompanied by reduced mRNA levels, suggesting that the KNAT1 mutations affected PIN2 expression at the posttranscriptional level. In vivo visualization of intracellular vacuolar targeting indicated that vacuolar degradation of PIN2–GFP was significantly promoted in the root tips of the bp allelic mutants. Together, these results demonstrate that KNAT1 negatively modulates root skewing, possibly by regulating auxin transport.
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Roots of higher plants change their growth direction in response to various environmental stimuli, such as gravity, light, moisture, nutrients, temperature and obstacles, to adapt to their environment and gain maximal advantages for growth (Mochizuki et al., 2005). When grown on hard-agar plates, especially if tilted from the vertical position, plant roots do not grow straight, but instead show a ‘waving’ or ‘skewing’ growth pattern (Oliva and Dunand, 2007). Formation of the waving growth pattern was considered to be due to the interaction between gravitropic and thigmotropic responses (Okada and Shimura, 1990; Thompson and Holbrook, 2004). The skewing pattern results from the interaction of anisotropic cell expansion, touch induction and microtubule orientation (Furutani et al., 2000; Hashimoto, 2002; Nakajima et al., 2004; Sedbrook et al., 2004). Although these complex patterns may be caused by forces from circumnutation, positive gravitropism, and thigmotropism (Darwin and Darwin, 1880; Okada and Shimura, 1990; Simmons et al., 1995a,b; Johnsson, 1997), how gravity regulates the waving or skewing of roots and whether gravity is crucial for these complex growth patterns still remains unclear.
To clarify the mechanism underlying root waving and skewing at the molecular level, several Arabidopsis mutants that presented changes in the direction of root growth have been isolated. Root-skewing mutants, including lefty1, lefty2, spr1, spr2 and wvd2, are mainly involved in the arrangement of cytoskeletal structures in epidermal cells (Furutani et al., 2000; Thitamadee et al., 2002; Sedbrook et al., 2004; Perrin et al., 2007). In addition, Oyama et al. (1997) noticed that a mutation in HY5, which encodes a basic leucine zipper transcription factor, could also trigger root skewing to the left.
The KNOTTED1-like homeobox (KNOX) genes regulate various aspects of development in all green plant lineages and play a key role in maintaining the shoot apical meristem (SAM) (Hay and Tsiantis, 2009). In addition to their functions in the SAM, there is also growing evidence that implicates the involvement of the Class I KNOX (KNOXI) genes in definition of inflorescence architecture (Lincoln et al., 1994; Douglas et al., 2002; Venglat et al., 2002). Maize RS1 and KNOX4 (Kerstetter et al., 1994), Arabidopsis KNAT1 (Truernit et al., 2006), KNAT2 (Hamant et al., 2002) and KNAT6 (Dean et al., 2004) are expressed in roots. However, the function of KNOXI genes in root development is still unclear. Arabidopsis KNAT1 (At4 g08150), also named BREVIPEDICELLUS (BP), belongs to the KNOXI gene family, which play a role in SAM maintenance (Douglas et al., 2002; Venglat et al., 2002). The mutant alleles of KNAT1 (bp-1, bp-2, bp-3 and bp-4), in the Landsberg erecta (Ler) background, are deficient in node, internode and pedicel anatomies, and defects in bp-5 (Columbia background) are less severe due to the presence of a wild-type ERECTA gene (Douglas et al., 2002). In the present study, we found that KNAT1 negatively regulates root skewing in Arabidopsis, and the reasons behind this phenomenon were explored.
KNAT1 suppresses root skewing
When grown vertically or inclined at 60° on the surface of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) that contained 0.8% agar, the roots of Arabidopsis wide-type Landsberg erecta (Ler) seedlings grew with a slight rightward skew when viewed from the bottom of the plate (Figure 1b,f). The roots of bp-1 mutant seedlings (Ler background) exhibited exaggerated rightward slanting with significant enlarged skewing angles (Figure 1d,h–j) under the same conditions. Because bp-1 was generated in the Ler background, which contains a mutation in the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER), we determined whether BP and ER functioned together in regulating skewing. We crossed bp-1 (bp/er) with wild-type Landsberg (Lan; Figure 1a,e) and generated the bp/ER genotype. Roots of the bp/ER mutant also exhibited exaggerated skewing to the right as had bp-1 roots under the same conditions (Figure 1c,g,i, j). Moreover, there was no significant difference in the skewing angles between Ler (BP/er) and Lan (BP/ER) roots grown either vertically or inclined at 60° (Figure 1i,j). This result indicates that BP alone regulated root skewing.
To further confirm the function of BP in regulation of root growth, we examined the behavior of the bp-5 mutant (an allele of bp-1) roots on inclined agar medium. The bp-5 roots also skewed to the right in comparison with wild-type Col roots (Figures 2a and S1), but the degree of skewing of bp-5 roots was less than that of the bp-1 roots (Figure 2a,c). The variation of root skewing between these two allelic mutants might be due to the difference in genetic background (Vaughn and Masson, 2011). In addition, root growth in transgenic seedlings that over-expressed the BP gene (p35S::BP) was similar to that of wild-type Col roots (Figures 2a and S1). Reverse transcriptioni polymerase chain reaction (RT-PCR) analysis revealed no KNAT1 transcript in the bp-1 and bp-5 mutant lines (Figure S2), a finding that indicated that they were indeed null mutants. These data suggest that BP negatively regulates root skewing to the right of the gravity vector on vertical and inclined agar surfaces.
Cell file rotation
Several studies have indicated that right-slanting root growth is accompanied by a characteristic rotation of the root tip (Simmons et al., 1995a,b; Rutherford and Masson, 1996), which can be traced to the cell file rotation (CFR) of the root epidermis (Okada and Shimura, 1990; Mochizuki et al., 2005; Pandey et al., 2008) and dynamic properties of cortical microtubules in the cells (Hashimoto, 2002; Mochizuki et al., 2005; Ishida et al., 2007). To observe any effect of the bp-1 and bp-5 mutations on root-tip rotation, we quantified the angles of root skewing (α) and CFR (β) in the bending region between the elongation and the maturation zone of the roots grown on 0.8% agar medium with or without 3 μm propyzamide (Figure 2). In the absence of the drug, the mutant bp-1 and bp-5 roots developed a more severe right-slanting phenotype than their respective wild-type Ler and Col roots (Figure 2a,c). In these right-skewing bp-1 and bp-5 roots, the epidermal cell files showed a left-handed helix in contrast with the relatively straight epidermal cell files in the wild-type Ler and Col roots (Figures 2b and S3a,b).
In the presence of propyzamide (3 μm), the angles of rightward skewing in the bp-1 and bp-5 mutants and their respective wild-type Ler and Col roots were significantly enhanced, accompanied with the increase in left-handed CFR (Figure 2b,c,d). However, the up-regulated intensity of root skewing and CFR caused by propyzamide is not synchronous. The root skewing of mutant bp-1 and wild-type Ler roots in response to propyzamide was indistinguishable, while the increase in CFR in the wild-type Ler roots was obvious compared with bp-1 roots under the same condition (Figure S3c,d). In contrast, changes in CFR caused by propyzamide between the bp-5 mutant and wild-type Col was not qualitatively distinct, while the average skewing angle of bp-5 roots increased significantly compared with Col (Figure S3c,d). In addition, responses of p35S::BP roots were not significantly different relative to that of Col in the absence or the presence of propyzamide (Figure 2a,c).
Lateral gravitropic correction
Because gravitropism is considered as a central principle in all models of root waving and skewing on inclined plates (Okada and Shimura, 1990; Simmons et al., 1995a,b; Mullen et al., 1998; Migliaccio and Piconese, 2001), we compared the response to gravistimulation of bp-1 roots with Ler roots. As shown in Figure 3(a), bp-1 roots exhibited a slower reorientation upon gravistimulation within the first 2 h, then followed by similar rates of curvature as Ler roots, but overall gravitropic curvature angles of the bp-1 roots were significantly smaller than those of Ler roots (Figure 3a). In addition, the difference in elongation growth rates of bp-1 and Ler roots was not apparent under the same conditions (Figure S4). To further dissect whether the right-slanting growth of the mutant bp roots was a result of lateral gravitropic correction, bp-1 and Ler seedlings were germinated and grown on a three-dimensional (3-D) clinostat, which induces constant random gravity stimulation (Hoson et al., 1996). Roots of both rotated bp-1 and Ler seedlings exhibited random growth patterns and did not grow to the right (Figure 3b). These results suggest that gravitropism is needed for the development of a right-skewing behavior of bp mutant roots.
Next, the roots of both bp-1 and Ler with distinct right-slanting phenotypes were chosen and rotated to make the root tips align with the gravity vector (Figure 3c). If root skewing is dependent on lateral gravitropic correction, these roots should skew to the right again. If not, these roots should grow along the gravity vector. As mentioned above, both bp-1 and Ler roots exhibited right-hand skewing on 60° inclined plates, but the skewing angles of bp-1 roots were greater than that of Ler roots (noted first gravistimulation under this condition in Figure 3c). Then we rotated the plates to make the root tips realign with the gravity vector (second gravistimulation). After 2 days incubation, both bp-1 and Ler roots skewed to the right again, but the slanting degrees of bp-1 roots were obviously larger than those of Ler (Figure 3c,d). The results of the third gravistimulation treatment was similar to the second gravistimulation (Figure 3c,d). At the fourth and the fifth gravistimulation treatments, the difference between bp-1 and Ler roots were significant, of which the bp-1 roots continued to skew to the right, but the Ler roots grew vertically along the gravity vector and did not skew (Figure 3d,e). Based on these observations, we concluded that the enlarged right-handed slanting of bp-1 roots might be related to the decrease of lateral gravitropic correction.
Auxin response and transport
Several hypotheses have suggested that auxin might be involved in regulating lateral gravitropic correction in root skewing and waving (Luschnig et al., 1998; Marchant et al., 1999). Thus, we investigated whether auxin was an important factor for eliciting increased root slanting of the bp mutants. Firstly, the sensitivity of bp roots to auxin was examined. Seeds were first germinated on hormone-free agar medium, then transferred and incubated on agar plates that contained various concentrations of indole-3-acetic acid (IAA) for an additional 3 days as described by Müller et al. (1998). The results revealed that elongation of the bp-1 roots was more sensitive than Ler roots at low concentrations of IAA (P < 0.0001, at 5 × 10−9 and 10−8m, respectively; Figure 4a). Similar responses were observed in bp-5 roots (P < 0.01, at 5 × 10−9 and 10−7m; Figure 4b). We then tested the effect of 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of auxin transport, on root skewing. Figure 4(c) shows that the continued skewing of bp-1 roots, which we had described above (Figure 3c), was completely inhibited when treated with 5 μm TIBA. These results suggest that skewing of the bp mutant roots might require normal auxin transport.
In roots, basipetal IAA movements (from the root tip toward the base of the root) have been suggested to control root elongation and gravity response (Petrάšek and Friml, 2009). We found inhibition of acropetal IAA transport (from the base toward the root tip) by application of TIBA at the root–shoot junction did not affect the skewing of bp-1 roots (Figure S5a), while inhibition of basipetal IAA transport by local application of TIBA abolished the bp-1 root skewing (Figure S5b).
PIN2 plays an essential role in the basipetal transport of auxin in Arabidopsis root tip (Chen et al., 1998; Rashotte et al., 2000, 2001). To further investigate if the effect of BP on root skewing is associated with the basipetal IAA transport, we crossed bp-5 with a basipetal auxin transport impaired mutant pin2 and obtained a bp-5/pin2 double mutant (Figures 5a and S6). On the surface of 60° titled agar, the double mutant bp-5/pin2 roots exhibited more counterclockwise coiling in comparison to the right-handed skewing bp-5 roots (Figure S6a,b). This coiling phenotype was also observed in pin2 roots under the same condition (Figure S6c), but the frequency of coiling roots in the bp-5/pin2 seedlings was significantly higher than those from pin2 seedlings (Figure S6e). The root-coiling behavior was considered as loss of lateral gravitropic correction in the roots (Thompson and Holbrook, 2004). On the surface of horizontal agar medium, both bp-5 and Col roots formed counterclockwise curling (Figure 5a), but the average diameter of coils in the bp-5 roots was significantly smaller than that of its wild-type roots (Figure 5c). Double mutant (bp-5/pin2) roots exhibited significantly reduced curling morphology by fewer numbers and larger diameters relative to that of bp-5, like the curling behavior of the pin2 roots (Figure 5b,c). Root curling of bp-5, pin2, bp-5/pin2 and Col were abolished when treated with TIBA (Figure 5a). These results indicated that the effect of BP on root skewing might be related to PIN2-mediated auxin transport, which could be blocked by TIBA.
In Arabidopsis roots, the auxin-responsive reporter DR5–GUS has been used to indicate indirectly the distribution of auxin (Hou et al., 2004). Our data showed strong GUS staining in Ler root tips, which appeared in the quiescent center and the central root cap cells, including columella initials and mature columella cells (Figure 6e), as reported by Sabatini et al. (1999). In contrast, bp-1 roots exhibited a high increase in GUS activity in the stele and all root cap cells (Figure 6a). This result indicated that a larger amount of auxin might be blocked in the bp-1 mutant root tips. To confirm this idea, basipetal auxin transport in both the bp-1 and Ler roots was quantified using auxin-induced beta-glucuronidase (uidA or GUS) gene expression back from the root tips. Ler and bp-1 seedlings with the auxin-induced DR5–GUS reporter were treated with 5 μm exogenous IAA applied by placing an agar droplet at a root tip and incubating for 1–6 h, then GUS expression was visualized histochemically (Figure 6b–d, f–h). The result showed that the distance of DR5–GUS expression from the root tip was decreased in bp-1 roots in comparison to those from Ler (Figure 6i). To further confirm this result, auxin transport was also measured using the 3H-IAA assay. The result also confirmed that auxin transport in bp-1 roots was significantly reduced in comparison with those in Ler roots, and this difference between the bp-1 and Ler roots was abolished by TIBA (Figure 6j).
Vacuolar degradation of PIN2-GFP
Basipetal auxin transport was found to be dependent on the level of PIN2 proteins at the plasma membrane (PM) of root-tip cells (Paciorek et al., 2005). To elucidate whether the alteration of basipetal auxin transport in the bp mutant roots was related to the function of PIN2, transgenic lines expressing pPIN2::PIN2–GFP were introgressed into the mutants bp-1 and bp-5 by crossing. As shown in Figure 7, the abundances of GFP signal at the PM of the epidermal cells of both the bp-1 and bp-5 roots were significantly lower than that of their wild-type counterparts (Figure 7a–d). This finding was consistent with the immunoblot analysis of PIN2–GFP fusion protein levels, which indicated that the expression of PIN2 protein in the bp-1 and bp-5 roots was significantly down-regulated in comparison with wild-type roots (Figure 7e). However, quantitative real-time PCR analysis demonstrated that the expression of PIN2 in bp-1 and bp-5 roots had no significant changes in comparison with the wild-types (Figure 7f,g). Previous studies have suggested that the level of PIN2 in the PM is highly dependent on posttranslational control, including protein degradation in lytic vacuoles (Kleine-Vehn and Friml, 2008). Thus, we assumed that BP might be involved in regulating PIN2 levels in the PM through posttranslational control. We examined the behavior of PIN2–GFP in root cells, which is already well understood with regard to its intracellular trafficking and vacuolar turnover (Kleine-Vehn and Friml, 2008). Vacuolar targeting of PIN2–GFP was assessed in the Ler and bp-1 mutants treated with 1 μm concanamycin A (ConA), which blocks protein degradation in the vacuole by inhibiting vacuolar H-ATPase activity and vacuole acidification (Pali et al., 2004). Our data indicated that most of the PIN2–GFP in the bp-1 root epidermal cells appeared in vesicles (likely lytic vacuole compartments) and decreased at the PM after treatment with ConA (Figure 8a,b,d). The intensity of PIN2–GFP in these vesicles per cell also increased in the bp-1 mutant compared with Ler (Figure 8c). bp-5 also showed a similar phenotype when treated by ConA (Figure S7). Taken together, we suggest that the decrease of PIN2 levels at the PM in bp-1 and bp-5 root-tip cells is the result of increased vacuolar targeting of PIN2.
In this study, the function of KNAT1 in controlling root skewing was analyzed and the findings are as follows. First, KNAT1 negatively modulates the skewing of Arabidopsis roots on the vertical and inclined agar surface. Secondly, the right-skewing of bp mutant roots are gravity and auxin transport dependent. Finally, the KNAT1 mutation resulted in decreased basipetal auxin transport and increased vacuolar degradation of PIN2 in the root.
A role for KNAT1 in root skewing
The KNOXI genes in Arabidopsis have been proven to play roles in root development (Serikawa et al., 1996, 1997; Hamant et al., 2002; Dean et al., 2004; Truernit et al., 2006). Transcript levels of the KNAT1 gene were found at very low levels in Arabidopsis roots under normal culture (Truernit et al., 2006), but could be up-regulated transiently in both root tips and branch point cells of the inflorescence stem by altered gravistimulation (Kimbrough et al., 2004; Wei et al., 2010). A gene expression map of the Arabidopsis root showed that KNAT1 is relatively highly expressed in the endodermis and the base of lateral root cells that cover the elongation zone and lower part of the differentiation zone (Birnbaum et al., 2003). Using a transgenic line that expressed the GUS gene driven by the KNAT1 promoter, we also found that the KNAT1 gene mainly expressed at the base of lateral roots and the pericycle and the endodermis in the elongation and the differentiation zones of the main roots, and slightly in root-tip region (Figure S8). The region of the root showing enhanced CFR in the bp-1 and bp-5 seedlings correlated with a skewing phenotype in the elongation and maturation zone, in which a high expression of KNAT1 in the wild-type roots was observed (Figures 2, S3 and S8). However, KNAT1 mainly expresses in the pericycle and the endodermal cell, how it regulates the CFR in the epidermal cells is still an open question. CFR in roots was considered closely related with the dynamic properties of cortical microtubules (Ishida et al., 2007). Our data showed that the increase of root skewing bp-1 and bp-5 seedling in the presence of microtubules destabilized drug was not synchronous with the change of CFR. Thus we could not conclude that the role of KNAT1 in regulating skewing of root tip was directly connected with the array of cortical microtubules in the epidermal cells. Buer et al. (2003) indicated that CFR was not essential for root skewing to occur and differential flank growth could also account for root skewing. That the skewing of bp mutant roots was gravity dependent and was strongly associated with the auxin transport (we will discuss in the following) indicating that KNAT1 might be involved in controlling the signaling pathway between the root tip and the skewing region.
How does gravity affect root skewing?
The roots of Arabidopsis seedlings grown on inclined agar surfaces exhibit waving and skewing behavior. Although the precise mechanisms of these growth patterns are still not well understood, both gravity and the contact between the medium and the root are considered as the major factors that determine these patterns (Oliva and Dunand, 2007). Rutherford and Masson (1996) elucidated that the right-slanting root of wild-type and the sku mutant was not caused by diagravitropism or an alteration in root gravitropism. Piconese et al. (2003) suggested that root skewing was the main consequence of circumnutation. Several previous space flight experiments revealed that roots exhibited random growth in microgravity (Johnsson et al., 1996; Mortley et al., 2008). However, a recent study performed on board STS-131 showed that the roots of space-grown Arabidopsis seedlings did not exhibit random growth, but skewed towards one direction, which suggested that an endogenous response in plants caused the roots to skew and that this default growth response was largely masked by normal 1 g conditions on Earth (Millar et al., 2011).
There are still many debates about whether gravity is the main factor that affects root skewing. We found that the roots of both bp-1 and Ler grown under the 3-D clinorotational condition grew randomly (Figure 3b). This result indicated that the right-slanting of bp-1 roots could be gravity dependent. The effects of gravitropism can be partially counteracted under the 3-D clinostat rotational condition, but the influence of gravitropism and thigmotropism on the roots caused by normal gravity still not be excluded. To further confirm whether root skewing is gravity dependent, the culture plates were rotated for five successive times to alter gravistimulation on the bp-1 and Ler roots. From the first to fifth rotation (Figure 3c,d), the effect of touch stresses between the roots and the medium was similar. Thus, this experiment allowed us to analyze the effect of gravity in regulating root skewing and exclude the interference of negative thigmotropism to a certain extent. The slanting angle of bp-1 roots was much larger than that of Ler from the first to third rotation. At the fourth and fifth rotation stages, bp-1 roots still strongly skewed to the right, while those from Ler grew almost in the direction of the gravity vector and not to the right. In addition, bp-1 roots exhibited decreased gravitropism in comparison with Ler roots. At present we are unclear as to whether the exaggerated skewing behavior of bp-1 mutant roots on agar medium is related to reduced gravitropism, but there is no doubt that the skewing of bp-1 mutant roots is gravity dependent.
Is KNAT1 involved in regulating auxin transport?
Recent evidence has shown that the KNOX transcription factors might control the development of meristematic tissues by balancing the activities of multiple hormones (Hay et al., 2004). A possible feedback relationship between KNOX proteins and auxin is indicated by data suggesting that KNOX proteins may inhibit auxin transport and act upstream it (Tsiantis et al., 1999; Treml et al., 2005). However, ectopic accumulation of KNOX protein in aberrant leaves that develop from shoot apical cultures treated with polar-auxin-transport inhibitors indicated that auxin-dependent processes acted upstream of KNOX gene regulation (Scanlon, 2003). The regulatory relationship between auxin and the KNOX genes is still unclear. Our data show that the enhanced root-skewing phenotype in bp mutant roots is the consequence of down-regulation of basipetal auxin transport and is not of the acropetal auxin transport (Figure S5). In addition, down-regulation of basipetal auxin transport caused an apparent accumulation of auxin in the bp mutant root-tip cells as evidenced by DR5–GUS expression.
Using a PIN2–GFP reporter along with western blot analysis, we found that this alteration in auxin polar transport was accompanied by a decrease in PIN2 signal intensity in the root-tip cells. Meanwhile, this reduction in PIN2 levels in the mutant root-tip cells was not accompanied by decreased mRNA levels, suggesting that the KNAT1 mutations affected PIN2 expression at the posttranscriptional level. Treatments with ConA that affects protein targeting to the vacuole resulted in enhanced PIN2 accumulation in the vesicles of bp mutants relative to the wild-type, suggesting that the KNAT1 mutation enhances vacuolar degradation of PIN2–GFP. We further examined the concentration of auxin in the tip regions of the bp-1 and wild-type roots and found a higher concentration of auxin accumulated in the bp-1 root tip (Figure 6a), where the increase in degradation of PIN2 was also observed (Figures 7 and 8). According to previous studies, the degradation of PIN2 would be induced when the concentration of auxin in the root-tip region was higher than a certain threshold (Paciorek et al., 2005; Abas et al., 2006). We assumed that the elevation of vacuolar degradation of PIN2 in bp mutant root-tip cells might be attributed to the retention of auxin in that region.
Several recent studies indicated that the endodermis and the pericycle in Arabidopsis roots might help to separate the acropetal and basipetal auxin fluxes in the stele and the epidermis, respectively (Blakeslee et al., 2007; Wu et al., 2007; Mravec et al., 2008). A special class of auxin transporters expressing in the endodermis and the pericycle are suggested to play a supportive role in controlling how much auxin is available for each PIN-base transport flow by recycling some auxin from the epidermis back to the vasculature (Blilou et al., 2005). The expression of the pBP::GUS reporter gene was mainly observed in the pericycle cells and the endodermal cells in the elongation and differential zone, but did not show much expression in the distal elongation zone and the root apex, where auxin is primarily accumulated (Figure S8). This raises a question on how to reconcile the differences between the site of BP action and expression. We currently cannot reach conclusions about the relationship between the auxin accumulation in the root tip and a mutation of KNAT1 in the root. In the future, the analysis of the interaction between KNAT1 protein and auxin transporters in the pericycle and endodermis will be crucial for understanding if the effect of KNAT1 in controlling root skewing is related with the regulation of auxin transport in the root.
Arabidopsis thaliana wild-type ecotype Columbia (Col), Landsberg erecta (Ler) and Landsberg ERECTA (Lan), mutant brevipedicellus (bp) bp-1 and bp-5 (Douglas et al., 2002), pin2 (Luschnig et al., 1998), and transformants proBP::GUS (Ori et al., 2000) and pro35S::BP (Lincoln et al., 1994) have been previously described. The single mutant of bp/ER genotype in Ler background was generated by crossing bp-1 (Ler background) with Lan. The F2 population in bp/ER phenotype was selected as described by Douglas et al. (2002). The bp-5/pin2 double mutant was generated by crossing bp-5 (Col background) with pin2 (Col background). The pPIN2::PIN2–GFP and DR5–GUS transformed seeds in the Columbia background were kindly provided by Dr Klaus Palme (University of Freiburg, Germany) (Blilou et al., 2005). The pPIN2::PIN2–GFP and DR5–GUS reporter genes were introduced into the bp-1 and bp-5 mutants, respective, by genetic crossing as described by Shin et al. (2005). Briefly, bp-1 [DR5–GUS] line was derived from a cross between bp-1 and DR5–GUS plants. F3 progeny homozygous for bp-1 and DR5–GUS reporter were used for experiments. Ler [DR5–GUS], bp-1 [pPIN2::PIN2–GFP], Ler [pPIN2::PIN2–GFP] and bp-5 [pPIN2::PIN2–GFP] were generated in the same way.
Plant growth and root-skewing assays
Seeds were surface-sterilized with 75% (v/v) ethanol for 1 min, followed by 2% (v/v) NaClO bleach with 0.01% (v/v) Triton X-100 detergent for 20 min. After five rinses with sterile water, seeds were germinated and grown on a medium containing MS salts (Murashige and Skoog, 1962), 1% sucrose (w/v) and 0.8% (w/v) agar in Petri dishes. The Petri dishes were kept in the cold (4°C) and dark for 2 days, then transferred to the growth chambers with 120 μmol s−1 m−2 fluorescent light in a 16 h-light/8 h-dark cycle at room temperature (22°C).
The root slanting assay was modified from the method described by Rutherford and Masson (1996). Briefly, seedlings were grown in the plates under the conditions described above and positioned at vertical and 60° titled angles. Images were taken 4 days after germination and analyzed with the Image J software (http://rsbweb.nih.gov/ij/). Similar strategies were used to quantify root gravitropism, except that vertical plates were rotated by 90° after 4 days of growth (Rutherford and Masson, 1996). Images of root tips were captured (Canon EOS 350D digital) at different times after reorientation. The angle of the root tip with respect to the gravity vector was measured from the pictures with Image J software.
In root curling assay, seedlings were grown vertically for 4 days on 1.2% agar MS medium as described above, then the plates were placed horizontally for additional 48 h to stimulate root curling. The 3-D clinostat experiment was carried out as described by Wei et al. (2010).
For assays involving IAA, TIBA (Sigma, http://www.sigmaaldrich.com) and propyzamide (Sigma), the compound was dissolved in ethanol or dimethyl sulfoxide (DMSO) and added to the molten media prior to pouring the plates. Auxin-resistance assays for roots of seedlings were determined as described by Müller et al. (1998). For ConA treatment, 5-day-old seedlings were transferred to medium containing 1 μm ConA and incubated for additional 8 h before being examined by microscopy.
Total RNA was extracted from the roots using an RNAiso plus Kit (TaKaRa, http://www.takara.com.cn) according to the manufacturer's instructions. cDNA was synthesized using 2 μg of total RNA and 100 U of ReverTra Ace reverse transcriptase (Toyobo) according to the manufacturer's instructions. Reactions of qRT-PCR were done in a 384-well plate format with 7900HT Fast Real-time PCR System (Applied Biosystems®, http://events-na.appliedbiosystems.com), and SYBR Green to monitor double-stranded DNA synthesis. The APT1 gene was used as reference for the PIN2 genes. The primers used in this study are listed in Table S1.
Laser scanning confocal microscopy
The GFP fluorescence was imaged under Olympus confocal laser scanning microscope (Olympus, FV1000, http://www.olympusamerica.com). For fluorescence quantification, the same confocal settings were used to compare GFP expression in the different genotypes/treatments. Image J was used to quantify fluorescence intensities.
Basipetal auxin transport assays by DR5–GUS auxin reporter and 3H-IAA
Auxin transport was monitored indirectly using the synthetic DR5–GUS auxin reporter in Ler [DR5–GUS] and bp-1 [DR5–GUS] seedling roots according to Lewis and Muday (2009).
DR5–GUS staining was used to estimate basipetal auxin transport by applying 5 μm concentrations of auxin in agar droplets to the root tips. Histochemical GUS staining was performed as described by Wei et al. (2010). Distance of the region of the root exhibiting auxin-induced GUS expression was measured with Image J. 3H-IAA assays were performed as described (Lewis and Muday, 2009) with minor modifications. An agar droplet containing 100 nm3H-IAA with or without 30 μm TIBA was applied at the root tip of 6-day-old seedlings and incubated for 6 h, then the apical 3 mm of the root was excised, and the amount of 3H-IAA in the subsequent 5 mm segment back from the root tip was collected and placed into 1 ml of scintillation fluid. The amount of radioactivity was determined after 18 h incubation using a Perkin Elmer 1450 Microbeta scintillation counter (Massachusetts, USA).
Western blot analysis
Roots of 6-day-old seedlings were harvested and homogenized in extraction buffer (0.5 m Tris–HCl, pH 7.4, 0.1 m PMSF). After heating for 10 min at 100°C, proteins (20 μg) were separated on a 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel. Immunoblots were carried out with a purified rabbit antibody raised against GFP and a horseradish peroxidase-conjugated secondary antibody.
The authors are indebted to Professor Hai Huang for providing bp-1, bp-5, proBP::GUS and p35S::BP seeds, Professor Hong-Xuan Lin for sharing [3H]-labelled IAA, Dr. Andy Tsun and Zhao Shan for critically reading the manuscript and Mr Xiao-Shu Gao for helping on confocal microscope analysis. This paper was supported by the National Basic Research Program of China (2011CB710902), the China Manned Space Flight Technology Project and the Strategic Pioneer Projects of CAS (XDA04020202).
Conflict of Interest
The authors declare that they have no conflict of interest.