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
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.