Growth and patterning are key processes that govern the development of multicellular organisms. In some cases, like early Drosophila
, these are independent. However, in many animals and plants, proper development frequently relies on tight coordination of growth and patterning. Disruption of this coordination can lead to unchecked cell growth, resulting in tumorigenesis or misshapen organs8
. Although the molecular mechanisms involved in pattern formation9–11
and in cell-cycle control12–15
have been well characterized independently, few connections have been made between the two16,17
. Here, we identify a new transcriptional regulatory relationship between two key regulators of organ patterning and specific components of the cell-cycle machinery.
SHR and SCR transcription factors control ground tissue patterning by regulating the formative asymmetric division in the immediate progeny of the ground tissue stem cells, known as cortex/endodermis initial (CEI) cells18,19
. SHR acts non-cell autonomously20–22
to activate the expression of SCR in the quiescent centre, CEI and endodermis5, 6, 23,24,25
. To gain insight into the role of the SHR/SCR network in controlling formative cell divisions, we expressed an inducible version of either SHR or SCR in its respective mutant background and characterized the timing of formative divisions after induction.
Before induction, SHR- and SCR-inducible plants had a single mutant ground tissue layer4,5
(Supplementary Fig. 1
). After SHR induction, SCR
expression was observed within 3 h, indicating that SHR rapidly activates its targets (Supplementary Fig. 1
). The first periclinal (parallel to the direction of growth) division in the mutant ground tissue layer occurred 6 h after SHR induction (, Supplementary Fig. 1
and Supplementary Movie 1
) and earlier after SCR induction (). Two layers of ground tissue with SCR
expression in the quiescent centre and endodermis (Supplementary Fig. 1
), along with a nearly complete Casparian band5
, were detected 24 h after SHR induction (Supplementary Fig. 2
). This underlines the combinatorial role of SHR and SCR in regulating formative cell divisions and also indicates that the two inducible systems have slightly different kinetics.
SHR and SCR regulate genes involved in formative cell divisions
To understand the dynamics of the SHR/SCR regulatory network, we sorted ground tissue cells at several time points after SHR and SCR induction and performed microarray analysis on RNA from the sorted cells26
. We found 2,478 and 1,903 differentially expressed genes throughout the SHR and SCR time courses, respectively (, Supplementary Tables 1 and 2
). Seven of the eight previously identified SHR direct target genes24,25
were differentially regulated in the SHR time course (Supplementary Fig. 3
) and most of them were also identified in the SCR time course (Supplementary Fig. 3
). For both inducible systems, the results for a subset of regulated genes were independently confirmed by quantitative PCR with reverse transcription (RT–qPCR) (Supplementary Fig. 3
We identified 12 clusters of co-expressed genes downstream of either SHR or SCR (Supplementary Fig. 4
). In both SHR and SCR time courses, known SHR targets were found in what we named the ‘SCR cluster’, as it included SCR
(Supplementary Fig. 5
). Notably, in the SCR-inducible system the expression level of these genes peaked at 3 h as compared to 6 h in the SHR-inducible system. The observed timing of the first formative division followed a similar progression. We therefore propose that genes in the SCR cluster may have a role in regulating formative cell divisions.
In each of the 12 clusters, we identified significantly enriched gene ontology categories (Supplementary Fig. 6, Supplementary Tables 3 and 4
). In clusters activated 1 h after SHR (cluster I) and SCR induction (clusters IV and V) (Supplementary Fig. 4
), transcription factors were significantly overrepresented. At 6 h after induction and coincident with the onset of formative divisions, gene ontology categories associated with cell-cycle progression and cyclin-dependent protein kinase (CDK) activity were overrepresented in both inducible systems (Supplementary Fig. 6
To examine the role of the SHR/SCR network in ground tissue formative divisions, we identified genes that were differentially expressed in both inducible systems. In total, 860 genes were regulated by both SHR and SCR ( and Supplementary Table 5
). We identified six clusters of co-expressed genes (Supplementary Fig. 7
), and gene ontology category enrichment analysis showed predominant molecular functions of these clusters ( and Supplementary Table 6
). Interestingly, one cluster (V) contained genes related to cell division and CDK activity. This indicates that SHR and SCR jointly regulate formative cell divisions by regulating cell-cycle machinery components.
To identify direct and indirect target genes of SHR, we performed chromatin immunoprecipitation-based microarray (ChIP-chip) experiments. We identified 266 genes as direct targets of SHR (Supplementary Table 7
). Of these, 65 were differentially expressed in the SHR-inducible system ( and Supplementary Table 8
). These genes were positively regulated by SHR and were found primarily in two major expression profiles, with activation at either 1 h or 6 h after SHR induction (Supplementary Fig. 8
). The proportion of transcription factor genes was significantly higher than expected by chance ( and Supplementary Table 9
), suggesting that SHR directly activates a regulatory cascade involved in several biological processes including cell-cycle progression. Coincident with the onset of periclinal divisions, we identified a D-type cyclin, CYCD6;1
, that was regulated and whose promoter was directly bound by SHR (). This gene was one of only six SHR direct target genes in the SCR cluster ( and Supplementary Fig. 9
). To confirm direct binding of SHR to the CYCD6;1
promoter and determine whether SCR also binds this target, we performed ChIP–qPCR. We found significant enrichment for both SHR and SCR binding around 1 kilo-base (kb) upstream of the CYCD6;1
promoter (Supplementary Fig. 10
). The temporal pattern of activation of CYCD6;1
in the SHR- and SCR-inducible systems, the binding of SHR and SCR to its promoter and the known protein–protein interaction of SHR and SCR25
support regulation of CYCD6;1
by both SHR and SCR.
SHR directly activates transcription factors and a cell-cycle gene
To determine if CYCD6;1
expression is consistent with a role in the formative division of the immediate progeny of the CEI, the CEI-daughter cell, we generated a transcriptional reporter for CYCD6;1
(pCYCD6;1::GFP). We observed its expression specifically in CEI/CEI-daughter cells (); however, we rarely detected expression in all eight CEI/CEI-daughter cells simultaneously, indicating that these formative divisions are not tightly synchronized. In cycd6;1
mutant seedlings (Supplementary Fig. 11
), CEI-daughter cells showed significantly fewer formative divisions (). Nevertheless, cortical cell number and overall meristem length were comparable to wild type (Supplementary Fig. 12
). These results indicate that CYCD6;1 is not required for proliferative cell divisions but is specifically involved in formative divisions needed for proper ground tissue patterning. Because CYCD6;1
is also expressed in embryonic CEI-daughter cells (), we examined ground tissue formative divisions during embryogenesis. cycd6;1
CEI-daughter cells showed significantly fewer periclinal divisions than wild type at both early heart and torpedo embryo stages, whereas all other embryonic root divisions appeared normal (). Interestingly, by the mature embryo stage the number of periclinal divisions in cycd6;1
and wild-type CEI-daughter cells was more similar (). We also observed no difference in the seed germination rate between wild type and cycd6;1
(Supplementary Fig. 13
and Supplementary Movie 2
). These results indicate that the cycd6;1
phenotype is specific to a small number of formative divisions and developmental stages.
Spatiotemporal activation of CYCD6;1
Later in root development, CYCD6;1
expression is restricted to a subset of endodermal cells () that undergo a second formative cell division to form middle cortex27
. In wild type, middle cortex divisions were observed in 52% of roots, as compared to 12% in cycd6;1
(data not shown). In older plants, expression of pCYCD6;1::GFP was detected in lateral root primordia () and pericycle and phloem cells (data not shown). Each of these tissues undergoes formative divisions and all are within the SHR and/or SCR functional domains17,24
Because the cycd6;1
phenotypes are not identical, we proposed that there is functional redundancy in the regulation of this formative division. To test this, we analysed mutations in CYCD2;1
, other D-type cyclins downstream of SHR (Supplementary Table 1
). We did not detect abnormal CEI-daughter divisions in either mutant. The double mutants, cycd6;1cycd2;1
(Supplementary Fig. 14
), were not significantly different from cycd6;1
, suggesting a higher level of functional redundancy, which may include other CYCD
genes. Taken together, we provide evidence that the SHR/SCR transcriptional network exerts finely tuned control of the formative divisions within the CEI through the restricted spatiotemporal activation of a specific cell-cycle gene.
To identify further genes that participate in this formative division, we profiled transcripts expressed in CEI/CEI-daughter cells by sorting cells from plants containing pCYCD6;1::GFP. Of genes highly expressed in CEI/CEI-daughter cells (Supplementary Table 10
), 234 were also differentially regulated in the SHR- and SCR-inducible systems. Notably, 32 showed G2/M-phase-specific expression during cell-cycle progression28
(Supplementary Fig. 15
). On the basis of root-cell-type microarray expression data29
, these mitotic genes are expressed in the same domain as CYCD6;1
(), indicating that they are involved in tissue-specific cell divisions. Whether they are involved specifically in formative divisions or more generally in proliferative divisions remains an open question.
SHR and SCR activate cell-cycle genes for formative divisions
Recently, genes involved in formative cell divisions during lateral root formation have been identified17
, 12 of which are regulated by SHR and SCR and expressed in CEI cells ( and Supplementary Table 11
). Among these are two CDKs, CDKB2;1
, indicating that SHR/SCR regulation of these genes may have a role in formative divisions of ground tissue. To test this hypothesis, we ectopically expressed CDKB2;1
, as well as CYCD6;1
, specifically in the ground tissue. Although root length in these lines was comparable to wild type, we observed extra formative divisions in endodermal cells (, Supplementary Figs 16 and 17
). We also ectopically expressed CYCD5;1
and found no alterations in patterning, indicating that not every D-type cyclin is able to stimulate this formative division (Supplementary Fig. 18
). Interestingly, CDKB2;1
were not identified as direct targets of SHR, suggesting that they may be regulated by transcription factors activated 1 h after SHR and SCR induction. Taken together, our results indicate that CDKB2;1, CDKB2;2 and CYCD6;1 are involved in specific formative cell divisions downstream of the SHR/SCR network.
To assess whether CYCD6;1 and CDKB2;1 could function independently of SHR in promoting formative divisions, we ectopically expressed them in the shr mutant ground tissue. Both CYCD6;1 and CDKB2;1 were able to partially complement the shr formative division phenotype (27% and 25% of plants showed divisions, respectively) (), whereas root length was comparable to shr-2-J0571 (). This indicates that these genes have important roles in generating formative divisions downstream of SHR but that there are further genes involved in this process.
We identified a component of the cell-cycle machinery, CYCD6;1, as a key SHR/SCR downstream target, providing the first evidence of a direct molecular link between these key developmental regulators and a cell-cycle gene. We further showed that CYCD6;1 is expressed specifically at the time of the formative cell divisions regulated by SHR/SCR and that altering its expression results in formative division changes in both loss- and gain-of-function plants. Thus, tight spatiotemporal regulation of specific cell-cycle genes is required for proper root pattern formation.