The bHLH transcription factor SPATULA regulates root growth by controlling the size of the root meristem
© Makkena and Lamb; licensee BioMed Central Ltd. 2013
Received: 16 July 2012
Accepted: 12 December 2012
Published: 2 January 2013
The Arabidopsis thaliana gene SPATULA (SPT), encoding a bHLH transcription factor, was originally identified for its role in pistil development. SPT is necessary for the growth and development of all carpel margin tissues including the style, stigma, septum and transmitting tract. Since then, it has been shown to have pleiotropic roles during development, including restricting the meristematic region of the leaf primordia and cotyledon expansion. Although SPT is expressed in roots, its role in this organ has not been investigated.
An analysis of embryo and root development showed that loss of SPT function causes an increase in quiescent center size in both the embryonic and postembryonic stem cell niches. In addition, root meristem size is larger due to increased division, which leads to a longer primary root. spt mutants exhibit other pleiotropic developmental phenotypes, including more flowers, shorter internodes and an extended flowering period. Genetic and molecular analysis suggests that SPT regulates cell proliferation in parallel to gibberellic acid as well as affecting auxin accumulation or transport.
Our data suggest that SPT functions in growth control throughout sporophytic growth of Arabidopsis, but is not necessary for cell fate decisions except during carpel development. SPT functions independently of gibberellic acid during root development, but may play a role in regulating auxin transport or accumulation. Our data suggests that SPT plays a role in control of root growth, similar to its roles in above ground tissues.
The primary root of Arabidopsis thaliana has a simple and consistent organization of cell types . Roots are divided into three distinct tissue zones along the proximal-distal axis. The most distal area is the zone of cell division or meristematic zone. A zone of cell elongation occurs just proximal to the division zone and the zone of cell differentiation or zone of maturation is the most proximal . Within the root apical meristem (RAM), stem cells surround a group of four mitotically less active cells called the Quiescent Center (QC; ). The QC, together with its surrounding four types of stem cells (columella stem cells, epidermal/lateral root cap stem cells, cortex/endodermal stem cells and vascular stem cells), forms the stem cell niche . The RAM is established during embryogenesis. In Arabidopsis, the zygote divides asymmetrically to form an apical and a basal daughter cell. Three rounds of stereotyped cell divisions in the apical daughter cell give rise to the apical and central regions of the embryo whereas the transverse divisions in the basal cell make about 6-9 cells that make up the extra-embryonic suspensor. The uppermost cell of the suspensor becomes the hypophysis, which divides transversely to make an upper and lower hypophyseal cell. The upper hypophyseal cell forms the QC and the lower hypophyseal cell forms the columella stem cells and the central root cap. The rest of the RAM arises from derivatives of the apical cell [3–5].
A complex network of transcription factors regulates specification of the root stem cell niche. The AP2/ERF transcription factor-encoding genes PLETHORA1 (PLT1) and PLT2 are transcribed in response to auxin in the early basal embryo and are redundantly required for QC identity and stem cell maintenance . Ectopic embryonic expression of PLT1 and PLT2 can induce the formation of a RAM, including the QC and initial cells . The PLT genes are expressed in a gradient with maximal expression in the stem cell niche promoting stem cell identity and maintenance. Lower levels promote mitotic activity of stem cell daughter cells and low levels promote cell differentiation . Two GRAS family transcription factors, SHORTROOT (SHR) and SCARECROW (SCR), regulate both radial patterning and stem cell niche specification in the root [8, 9]. SHR is necessary both for the periclinal division of cortex/endodermal initials and endodermal specification [10–13]. SCR is required for the periclinal asymmetric division of the cortical/endodermal initial daughter cells and cell-autonomously required for QC identity [9, 14, 15].
The SPATULA (SPT) gene encodes a basic helix-loop-helix (bHLH) transcription factor and was originally identified for its role in carpel organogenesis . SPT is necessary for the development and proliferation of the carpel margins and for development of tissues derived from the margin [16, 17]. SPT is partially redundant with the closely related bHLH-encoding gene ALCATRAZ (ALC) and functions during fruit development to specify formation of the valve margin tissue in addition to margin tissues during pistil development. These proteins can heterodimerize and SPT can complement dehiscence defects in alc mutants if expressed appropriately, although the converse is not true . In addition to its interaction with ALC, SPT interacts genetically and physically with another bHLH protein, INDEHISCENT (IND), in both carpel margin and fruit valve specification and these transcription factors may bind common target genes cooperatively . SPT and IND proteins regulate auxin accumulation in the apical region of the developing carpels, important for carpel margin specification. This is due at least in part by their direct regulation of expression of two genes encoding members of the AGC3 family of protein kinases (PID and WAG2) that phosphorylate and control activity of PIN auxin efflux carriers .
Although SPT has been most extensively studied in the context of floral development, it has been shown to be involved in seed germination and leaf and cotyledon development [20–23]. SPT has also been shown to mediate vegetative growth repression in response to cool day temperatures  and spt mutants have larger leaves due to increased cell numbers and an enlarged meristematic region in leaf primordia  and larger cotyledons due to increased cell expansion . Consistent with its broad function, SPT is expressed in proliferating regions of both vegetative and reproductive tissues, including the root [16, 24].
Here, we investigate the role of SPT in root growth. spt mutants have longer primary roots due to an increase in cell proliferation. Examination of the RAM of these mutants showed that QC size is increased as is cell division in the initial cells, as measured by the cell division marker pCYCB1;1::GUS. The increased QC size arises in the embryo and extra divisions continue throughout root development. The effect of loss of SPT function on root growth is independent of GA but shares some common targets with this hormone. spt mutants show a larger auxin maximum at the root tip. Analysis of the mutants’ responses to exogenous auxin and auxin transport inhibitors suggests that auxin transport is likely to be altered. Control of auxin transport may be a common mechanism by which SPT regulates growth in the plant. Our results uncover the importance of SPT in the regulation of RAM size control.
SPTis necessary for multiple developmental aspects of plant development
SPT impacts plant growth a
Total plant heightb
Days to flower
Number of rosette leaves
Number of flowers
Number of internodes
Length of internodesb
22.4 ± 0.4
17.6 ± 0.6
49.1 ± 1.0
51.9 ± 1.0
0.57 ± 0.01
30.0 ± 0.7
23.6 ± 0.3c
27.1 ± 0.9c
74.5 ± 1.2c
78.0 ± 1.3c
0.42 ± 0.004c
35.0 ± 0.5c
29.9 ± 0.7
39.3 ± 1.4
71.7 ± 2.0
75.0 ± 1.9
0.83 ± 0.008
63.2 ± 1.6
28 ± 0.8
36.8 ± 1.5
80.8 ± 2.8c
84.0 ± 2.6c
0.77 ± 0.012c
65.8 ± 1.3
SPTcontrols the size of the quiescent center of both the embryonic and postembryonic stem cell niches
Loss of SPTfunction leads to a broader auxin maximum at the root tip but does not disrupt root patterning
Response to exogenous NAA is not changed by loss of SPT function
Concentration of NAA (nM)
Root length (mm)
% of mock
24.7 ± 6.7
17.1 ± 4.2
4.6 ± 1.1
3.4 ± 1.0
3.1 ± 0.7
2.7 ± 0.7
2.2 ± 0.6
26.2 ± 3.7
17.7 ± 3.0
5.2 ± 1.7
4.8 ± 1.6
3.5 ± 0.9
3.2 ± 1.0
2.7 ± 1.4
Loss of SPT function confers increased sensitivity to NPA
Concentration of NPA (μM)
Root length (mm)
% of mock
17.5 ± 3.3
9.5 ± 1.5
4.3 ± 1.0
3.6 ± 0.7
2.2 ± 0.6
23.4 ± 2.9b
8.0 ± 1.0b
4.6 ± 1.0b
3.8 ± 0.4b
2.7 ± 0.5
Root stem cell niche specification and radial patterning are regulated in part by two transcription factors, SHR and SCR [8–10, 37]. Since spt-11 has more cells in the QC (Figures 3, 4), we looked at the expression of SCR and SHR to see if they are disrupted. pSCR::GFP is expressed in the endodermal layer, endodermal/cortical initials and QC (Figure 5E; ) while pSHR::GFP is expressed in the stelar cells (Figure 5G; ) and pSHR::SHR-GFP is expressed in the stelar tissue, endodermal cell layer, endodermal/cortical initials and QC (Figure 5I; ). No differences in the expression domains of these genes were seen in spt-11 when compared to wild type seedlings (Figure 5F, H, J), indicating that SPT does not regulate stem cell niche positioning or radial patterning.
SPT expression has been detected in vascular tissues . In order to check if SPT has any role in the development of vascular elements, we looked at the expression of two vascular markers. Enhancer trap J0121::GFP is specifically expressed in the xylem-associated pericycle cells (Additional file 3A; ) and the marker CoYMV::GFP is specifically expressed in the phloem companion cells (Additional file 3C; ). The expression of these markers in spt-11 is similar to wild type expression patterns (Additional file 3B, D), suggesting that loss of spt does not disrupt differentiation of the vasculature. We also looked at whether the radial organization of the root is affected in spt-11. Col-0 roots have 8 cells in the cortical cell layer (Figure 5K; n = 15 seedlings; >20 sections/seedling) as previously reported , whereas spt-11 roots have 9 cells in the cortical cell layer (Figure 5L; n = 15 seedlings; >20 sections/seedling), likely reflecting the increased cell division in the root meristem. However, the overall organization of the root is unaffected.
SPTacts in parallel to GA in the root
To further investigate the relationship between SPT-mediated cell proliferation and GA, we crossed spt-11 and spt-2 mutants to GA biosynthesis mutants in the appropriate genetic backgrounds, ga3ox1-2; ga3ox2-1 and ga1-3, respectively. Root meristem size was examined in 7-day-old seedlings of Col-0, spt-11, ga3ox1-2; ga3ox2-1 and the triple mutant ga3ox1-2; ga3ox2-1; spt-11. The double mutant of ga3ox1-2; ga3ox2-1 has a short RAM, as previously reported (Figure 6H, J; ) while spt-11 has the longest RAM among all the genotypes analyzed (Figure 6G, J). The triple mutant ga3ox1-2; ga3ox2-1; spt-11 has a significantly bigger RAM than that of double mutant ga3ox1-2; ga3ox2-1, but a smaller RAM than spt-11 (Figure 6I, J), an additive phenotype. This triple mutant and the ga1-3; spt-2 double mutant were analyzed for other developmental differences. ga3ox1-2; ga3ox2-1; spt-11 plants as well as ga1-3; spt-2 plants are of intermediate height compared to their parents (Additional files 4 and 5). Triple mutants flowered significantly earlier than the ga3ox1-2; ga3ox2-1 plants, have significantly higher number of flowers and internodes, significantly longer internodes and are significantly taller (Additional file 5), suggesting that the loss of SPT function can partially compensate for lower GA levels in the plant. However, the triple mutant plants are neither equivalent to spt-11 nor wild type plants. This additive phenotype suggests that SPT and GA act in parallel pathways. The fruit phenotype of spt-11 is still retained in ga3ox1-2; ga3ox2-1; spt-11 plants (data not shown), suggesting either that GA is not functioning in apical carpel development or that SPT acts downstream of GA in this context.
Since the above expression results were inconclusive, we examined the expression of several downstream GA-responsive genes: SCARECROW-LIKE 3 (SCL3), which is downregulated in response to GA, and EXPANSIN1 (EXP1) and GIBBERELLIC ACID INSENSITIVE (GAI), both of which are increased by GA . Consistent with previous reports, exogenous GA does not significantly increase GA-induced gene expression in the L. er background (Figure 7D; ). In the absence of GA application, there was no significant difference in expression of SCL3, EXP1 or GAI between spt-11 or spt-2 and wild type (Figure 7C, D). Upon GA application, the significant increase in EXP1 expression seen in wild type is not observed in spt-11 (Figure 7C), perhaps suggesting that SPT contributes to regulation of this gene. However, similar to the situation seen with the GA biosynthetic and catabolic genes, no consistent pattern of GA responsive gene expression was seen.
Previous studies have shown that SPT functions in diverse organs of the aerial portion of Arabidopsis, including the cotyledon , the leaf , the gynoecium [16, 17, 57], the fruit [18, 19] and in germinating seeds . In this study we have shown that SPT also functions in the root, where it acts to restrict RAM size and root length. Loss of function spt mutants have a larger zone of cell division (Figure 1), which contains more dividing cells than wild type (Additional file 2); this leads to a higher growth rate in the roots and longer primary roots. In adult plants, the inflorescence stem is significantly longer than that of wild type and produces more flowers (Table 1). It has previously been shown that spt-11 plants have larger leaf areas due to increased cell number  and cotyledon size due to increased cell expansion . Taken together, these data suggest that SPT acts to restrict cell proliferation and expansion in a number of organs in Arabidopsis.
QC size was increased in both spt-2 and spt-11 roots, as assayed by morphology and molecular markers. In the wild type background, the QC consists of on average four cells that are mitotically less active than the surrounding initial cells . However, spt mutants have an increased number of cells in their QCs, often being three or four cells across instead of two and sometimes having two layers of QC cells (Figures 3, 4). The increase in size of the QC is evident in the embryo, starting at approximately torpedo stage (Figure 3). spt-11 embryos can have up to 6 cells in their QC and the size increase continues during root development, as roots with up to 10 QC cells were observed. The increase in the size of the QC and the root division zone in roots of spt mutants is similar to the increase in the size of the meristematic region of leaves in spt mutants , suggesting that the molecular pathway in which SPT functions may be similar in these two organs.
SPT expression is correlated with areas of high auxin content [16, 24], suggesting a relationship between SPT and auxin. The SPT promoter also contains several auxin response elements (AREs), suggesting that auxin response factors (ARFs) may directly regulate its expression. However, it has previously been shown that mutating these elements does not change expression of a SPT reporter . The increase in size of the RAM seen in spt mutants is correlated with a broader zone of expression of the auxin efflux carrier PIN4 and a stronger auxin maximum, as visualized by DR5::GUS expression (Figure 5). This may result from changes in auxin transport, as seen in developing carpels of spt mutants [19, 30], which is supported by the increased sensitivity to NPA shown by spt-11 roots (Table 3). spt carpel defects can be rescued by application of the auxin transport inhibitor NPA [30, 57], suggesting that SPT activity may impact auxin transport, which is consistent with its regulation of protein kinases that regulate the PIN efflux carriers . However, in the root, spt-11 mutants are hypersensitive to NPA application (Table 3) while in the carpel its application ameliorates the developmental defects caused by loss of SPT. A possible explanation for this could be differences between auxin transport in the carpel and the root tips. Carpel development depends on an auxin gradient along the apical-basal axis of the gynoecium, with the highest level of auxin present in the apex. Root development and growth, however, depends on both on an apical-basal auxin gradient with its greatest concentration in the region of the QC (generated by polar transport through protophloem cells) and redirection of that auxin flow laterally in the root cap where it subsequently flows back toward the shoot (through lateral root cap and epidermal cells) . Disruption of any of these auxin transport pathways impacts root growth and development [34, 59]. Thus, NPA application on the root tip likely causes more complex changes in auxin flow and accumulation compared to its effect in the carpel. SPT may regulate not only apical-basal auxin transport but also the auxin redirection pathway as well.
Our data, similar to that reported by other groups working on shoot organs , suggests that SPT functions in parallel to GA to regulate RAM size and root length. It has long been known that there is crosstalk between auxin and GA and between auxin transport and GA. Recently it has been shown that GA-deficient plants accumulate fewer PIN auxin transport proteins, although PIN4 accumulation was not evaluated, and that this correlates with less auxin transport . Therefore, it is possible that changes in GA content or signalling in spt mutants might lead to the changes in auxin accumulation at the root tip we observed. Clearly more work is necessary to determine the relationship(s) between GA, auxin and SPT.
As mentioned above, SPT has functions in germination, cotyledon expansion, leaf size and gynoecium development. Our work extends the functions of this gene into the root, where it acts to regulate cell proliferation in the meristematic zone without impacting overall root organization or differentiation in the mature area of the root. This is similar to the role of SPT in leaf growth control, where it appears to act by restricting the size of the basal meristematic zone of the leaf without altering leaf morphology or cell types . This is in contrast to the effect of loss of SPT in the flower, where less cell proliferation takes place in the gynoecium, resulting in a shorter pistil with defects in stigma, style and transmitting tract tissues . However, since SPT may act through regulation of auxin transport in both the carpel and root, the varying impacts on cell proliferation in these organs may be due to differences in auxin response.
SPT encodes a bHLH protein  that has been shown to act as a transcriptional activator . bHLH proteins act in dimers or larger order protein complexes. SPT belongs to a subclade of bHLH factors (Group VII of /subfamily 15 of ). This group has fourteen members of which the ALC gene, partially redundant with SPT[18, 64], is most closely related. These proteins can heterodimerize with each other ; however, ALC is not highly expressed in the root (Genevestigator; [65, 66]). In addition, the PIF/PIL bHLH proteins fall in this clade, which interact with phytochromes and contain a PHYB-binding domain not found in either SPT or ALC [63, 67]. SPT has been shown to interact genetically with PIL5 during seed germination  and PIL5 is known to regulate GA responsiveness . SPT also interacts with PIF6 during pistil development , suggesting it may be able to act with the products of these genes to regulate gene expression. However, root expression of these genes is low (Genevestigator; [65, 66]). SPT also heterodimerizes with members of the HECATE family of bHLH transcription factors . Loss of these genes causes carpel defects similar to those of spt mutants and they are expressed in an overlapping pattern with SPT in the carpel, but are not expressed in the root. Additionally, SPT interacts with IND in the carpel and fruit where it may bind DNA cooperatively with that protein ; it is unknown if this gene is expressed in roots.
While no bHLH proteins have been shown to interact with SPT in the root to date and its known interactors are not known to be expressed in this organ, at least three genes encoding bHLH transcription factors are active in root growth control. LONESOME HIGHWAY (LHW) regulates the size of the stem cell pool that gives rise to the cells of the root vascular cylinder , while UPBEAT1 (UPB1) regulates the expression of peroxidases to modulate the balance of reactive oxygen species between the zone of cell division and the elongation zone, regulating the onset of differentiation . Expression of both of these factors partially overlaps with that of SPT. In addition, MYC2 has been shown to be necessary for the jasmonate-mediated repression of root growth by directly repressing expression of PLT1 and PLT2. Examination of binding partners of SPT in the root and identification of target genes in this organ will provide great insight into the molecular pathway or pathways in which SPT acts.
SPT has previously been shown to regulate growth in several above ground organs of Arabidopsis. SPT also regulates proliferation in the root, controlling the size of the RAM and the number of cells in the QC. However, the organization of the root and differentiation of root cell types is not altered, although extra cells are made. SPT regulates growth in parallel to GA and by modifying the accumulation of auxin in the region of the QC, likely via regulation of auxin transport.
Plant materials and growth conditions
The spt-2 allele is in the L. er background and has been previously described . The spt-11 allele, a T-DNA insertion line in the Col-0 background, has been previously described  and is from the WISCDSLOX collection . Seeds of other mutants used in the study have also been described: ga1-3 and ga3ox1-3; ga3ox2-1. The double mutant ga1-3; spt-2 was generated by crossing homozygous ga1-3 and homozygous spt-2 plants and allowing the F1 to self-fertilize. The triple mutant ga3ox1-3; ga3ox2-1; spt-11 was generated by crossing homozygous ga3ox1-3; ga3ox2-1 plants and homozygous spt-11 plants and allowing the F1 to self-fertilize. Double and triple mutants were identified by PCR genotyping of the segregating F2 population. To generate spt-11 mutants containing marker lines, homozygous spt-11 plants were crossed to the lines (Additional file 6) and the F1 allowed to self-fertilize. Mutants were identified by PCR genotyping of the F3 after growth on antibiotic containing media to select for the transgene.
Arabidopsis thaliana seeds were cold treated for 3 days at 4°C and were germinated and grown on Fafard 2 mix soil (Fafard) under long-day (16 hours, 80 μmol m-2 s-1) irradiance, either in controlled growth chambers (Enconair Ecological Chambers Inc., Manitoba, Canada) or growth rooms with subirrigation at 22°C with 60% relative humidity.
Seeds used in all the assays done on seedlings were sterilized as previously described , placed on either on Murashige & Skoog (MS) media (Research Products International Corporation, Mt. Prospect, IL) with 1% plant agar in Petri plates or on half GM plates (half concentration of MS salts, 1% sucrose, 0.8% Plant agar, pH 5.7). The plates were incubated in the dark at 4°C for 5 days. The plates were then moved to a CU-36L growth chamber (Percival Scientific Inc., Perry, IA), placed vertically and grown under long day conditions as above unless noted.
Mutants were identified by PCR genotyping of genomic DNA. Genomic DNA was extracted from inflorescences and leaves as described previously . Primer sequences are shown in Additional file 7 and combinations used for genotyping various mutants are listed in Additional file 8.
In order to analyze various developmental phenotypes of mutants and wild type, plants were grown under long day conditions. Seeds of various genotypes were sown in square cells or pots in a randomized block design. After seeds were germinated, all seedlings except one per cell or pot were weeded out.
Several aerial phenotypes were analyzed as previously described . Leaf number at flowering was defined as the number of leaves when the first flower opened. In order to analyze root phenotypes, plants were grown on vertically oriented plates. For all the measurements done on seedlings in this study, the first day of incubation in the chamber was counted as day zero. Seedlings were collected for analysis at different Days After Germination (DAG) starting at 3 DAG till 11 DAG. To visualize roots using microscopy, seedlings were fixed overnight in ethanol and acetic acid (9:1). Roots were cleared in chloral hydrate (80 grams of chloral hydrate, 20 ml of water and 10 ml of glycerol) on microscope slides for 20 minutes for microscopic analysis with Differential Interference Contrast (DIC) optics on a Nikon Eclipse 90i. Pictures were taken using the attached Nikon camera and analyzed with NIS elements Advanced Research software version 3.0. Fifteen seedlings per genotype were used for root meristem size measurement. Root meristem size was measured as the number of cells in the cortical cell layer between the QC and the first elongating cell as described  and the results were depicted in graphical format using Prism (http://www.graphpad.com/prism/; GraphPad Software, La Jolla, CA). Results were analyzed statistically using a two-sample student t-test. The length of the meristematic zone was measured in micrometers from the QC to the first elongating cell in the root cortical cell layer as described . Results were analyzed and displayed as above.
In order to determine the effect of PAC (PhytoTechnology Laboratories, Shawnee Mission, KS) on root growth, seedlings were transferred from half GM plates to half GM plates with 10μM PAC or half GM plates with methanol 4 days after germination. The seedlings were grown in the incubator for 96 hours before they were analyzed for root meristem size as above.
The average primary root length was determined using 30 seedlings of each genotype per replicate with 3 replications. Measurements were taken at 7, 10 and 13 DAG. Primary root growth rate was measured by drawing a line at the tip of primary root on the back of the plate every day, starting from 2 DAG. The distance between the two markings was measured with a ruler. The lengths between the two time points were used for obtaining the growth rate per hour (length in mm/24 hours).
To look at the cellular organization of roots, 5-day-old roots were hand-sectioned according to the protocol “Rapid preparations of transverse sections of plant roots” (http://www.mcdb.lsa.umich.edu/labs/schiefel/protocols.html). The cross-sections were cut perpendicular to the length of the root beginning at the root tip and moving towards the base of the root. The root sections were transferred to a Petri dish containing fluorescent brightener 28 (FB 28 Sigma-Aldrich, St. Louis, MO) dissolved in water. Sections were stained for 10 minutes and examined using UV epifluorescence microscopy using a Nikon Eclipse 90i Microscope. 15 seedlings of each genotype were examined and at least 20 sections per seedling were analyzed.
To visualize embryos for microscopy, seeds containing embryos at different stages of development were collected from developing fruits and processed as previously described . Embryos were visualized with DIC optics on a Nikon Eclipse 90i. Pictures were taken using the attached Nikon camera and analyzed with NIS elements Advanced Research software version 3.0.
Gene expression studies
To examine SPT expression in roots, roots of 7 DAG L. er seedlings grown on plates were collected and stored at -80. Total RNA from two biological replicates was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to instructions. During the RNA purification, on column DNase treatment was done using RNase-free DNase (Qiagen) and RNA was also treated with DNase again in solution. 500 ng of RNA was used as template and cDNA synthesis and PCR was done using the SuperScript III One-Step RT-PCR System (Invitrogen by Life Technologies) using SPT and ACTIN-specific primers (Additional file 7). For real-time PCR analysis, the Blue Print First Strand cDNA Synthesis Kit (Takara Bio Inc., Otsu, Shiga, Japan) was used with 1 μg of total RNA as template to generate cDNA. 0.5 μl of cDNA was used in real-time PCR (qPCR) reactions done using the iQ™ SYBR Green Supermix (BioRad) on a CFX96™ Real-Time PCR detection system (BioRad) at the PMGF. A reference gene, At1g13320, was used to normalize the qPCR data . qPCR data was analyzed using CFX96 software and graphs were made using Prism. Primers for qPCR were designed using QuantPrime Q-PCR primer design tool (Additional files 7 and 9; http://www.quantprime.de; ).
For gene expression studies on GA response genes, seedlings were collected at 7 DAG and processed as above. Each genotype was represented by three biological replicates. For GA responsive gene expression, 7-day-old seedlings were treated with either 50 μM GA or mock in MS liquid media (1X MS salts, 1X Gamborg’s B5 Vitamins, 3% Sucrose with pH 5.7) for three hours in the incubator under constant light at 22°C. For quantifying GA biosynthesis and metabolism genes, three biological and two technical replicates were done. A reference gene, At1g13320, was used to normalize the qPCR data for GA biosynthesis and metabolism genes, while another reference gene, At4g33380, was used for normalizing the qPCR data for GA responsive genes . Primers were designed as above. Results were analyzed statistically using a non-parametric Wilcoxon rank sum test for GA biosynthesis, catabolism and receptor genes and using a 2-way analysis of variance for GA responsive genes [76, 77].
ß-glucuronidase and starch staining
Expression of QC25::GUS was examined as previously described . Photographs were taken using a Nikon Digital Sight DS-5M camera attached to a Nikon SMZ800 dissecting or on a Nikon Eclipse E200 compound microscope. GUS-stained 5-day-old seedlings were used for visualizing starch granule accumulation in the columella root cap cells. Staining for starch granules was done according to  in 1% lugol solution for 3 minutes, rinsed in water, cleared in chloral hydrate and photographed using Nomarski optics on a Nikon Eclipse 90i Microscope.
Confocal laser microscopy was used for looking at the expression of various cell specific markers tagged with Green Fluorescent Protein (GFP). The cell walls of various stages of embryos and roots were labelled with propidium iodide and were observed according to  with a Nikon D-Eclipse C1si Confocal.
NAA and NPA assays
NAA and NPA assays were done as described in . Briefly, seeds of Col-0 and spt-11 were sown on MS media with varying concentrations of NAA (0, 1, 20, 40, 60, 80 and 100 nM) or NPA (0, 0.1, 0.5, 1 and 2 μM). Seeds were cold treated for two days and then grown vertically as described above. Root length of 8-day-old seedlings was measured as described above. The average data from three independent experiments are presented and at least 20 seedlings were analyzed per genotype per experiment.
Root apical meristem
- L. er:
Gibberellic acid insensitive.
This work was supported in part by a grant from the National Science Foundation (MCD-0418891) to RSL and funds from The Ohio State University. The authors thank Dr. Patrice Hamel, Dr. JC Jang and Dr. Iris Meier (Ohio State University) and members of the Lamb laboratory for helpful advice on the manuscript and Rachel Edwards for technical assistance.
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