Repression of AS2 by WOX family transcription factors is required for leaf development in Medicago and Arabidopsis

Abstract

WOX transcription factors are key regulators of meristematic activity in plants. The Medicago WOX gene, STF, functions in maintenance of leaf marginal meristem, analogous to the function of WUS in the shoot apical meristem. Both STF and WUS directly repress AS2 expression in their respective domains. Ectopic expression of AS2 with WUS promoter leads to a narrow leaf phenotype and other phenotypes similar to the wus mutant. We also found that a wox1 prs wus triple mutant produces much narrower leaf blades than the wox1 prs double mutant, indicating that WUS genetically interacts with WOX1 and PRS in Arabidopsis leaf blade development. Our data points to a general requirement for AS2 repression in meristematic regions to allow cell proliferation.

The organogenesis of multicellular organisms needs the coordination of cell proliferation, differentiation and expansion.^1,2 Stem cell proliferation provides sufficient cell number needed for a specific tissue, while cell differentiation specifies various cells with distinct characteristics, and cell expansion results in cells with appropriate size to function as a tissue. Thus, stem cell proliferation and its maintenance is a prerequisite for development of multicellular organisms. In plants, stem cell maintenance is governed by plant-specific homeodomain transcription factors called WUSCHEL-related Homeobox (WOX) family proteins, named after the founding member WUSCHEL (WUS).³ In Arabidopsis, different WOX family members are responsible for the maintenance of meristematic cell activity in the tissues that they are expressed. For example, WUS is required for maintenance of the shoot apical meristem, SAM;³ WOX5 for root apical meristem, RAM;⁴ and WOX4 for vascular meristem, procambium.⁵ Leaf blade lateral development and outgrowth is controlled at the juxtaposition between adaxial and abaxial cell fates, a meristematic domain in the leaf (designated as Leaf Marginal Meristem, LMM).^6-11

In monocots, this meristematic domain is maintained by the WOX3/PRS homologues, named NARROWSHEATH 1 and 2 (NS1 and NS2) in Zea Mays,¹² and NARROW LEAF2 and NARROW LEAF3 in Oryza sativa.^13,14 In several other sequenced monocot-species, such as Musa acuminata, Sorghum bicolor, Brachypodium distachyon and Phyllostachys heterocycla, WOX3 homologues are all duplicated one or more times, suggesting their important roles in monocot plant development. In dicots, however, WOX3 usually exists as a single copy in the genome. Recently, several groups reported that the LMM in several dicot-species is instead mainly regulated by WOX1 homologues, including MAEWEST (MAW) in Petunia x hybrida,¹⁵ LAM1 in Nicotiana sylverstris,¹⁶ STENOFOLIA (STF) in Medicago truncatula,¹⁶ and LATHYROIDES (LATH) in Pisum sativum.¹⁷ In all of these species, single loss-of-function mutants in WOX1 homologues produce severely affected narrow leaf blades and floral organs with the exception of Arabidopsis thaliana, in which WOX3 functions redundantly with WOX1 in leaf blade outgrowth.^15,18 WOX1 genes are found in dicots and the basal angiosperm plant Amborella trichopoda, but not in monocot species sequenced thus far.^15,19-21

We previously showed that STF promotes cell proliferation to modulate leaf blade outgrowth in M. truncatula.¹⁶ STF mainly acts as a repressor and an evolutionarily conserved WUS-box is required for its repressive activity and biological function.²² More recently, we further dissected the STF functional domains and uncovered that the Homeodomain and the C-terminal domain (CTD) are critically required for STF function.¹⁹ Further analysis showed that STF recruits MtTPL through the WUS-box and STF-box in the CTD¹⁹ and this interaction provides the repressive activity required for cell proliferation in leaf blade outgrowth. Protein-protein interaction between WUS and TPL was shown to be important for floral development²³ and stem cell maintenance in the SAM.²⁴ Whether other WOX-TPL interactions are important for the stem cell maintenance in their respective domains such as the RAM and the vascular meristem is yet to be shown. However, TPL is known to mediate auxin signaling through direct interaction with ARFs and AUX/IAAs,²⁵ and the functions of WOX2 in the embryo,²⁶ WOX4 in the vascular meristem²⁷ and WOX5 in the RAM^28-30 have been associated with auxin signaling. It is, therefore, likely that these auxin signaling mediated functions could be modulated through interaction with TPL although direct experimental evidence is lacking.

One Important Direct Target of STF, AS2, Is Also Targeted by WUS

The key function of STF is to promote cell proliferation at the boundary between adaxial and abaxial domains, generating sufficient undifferentiated cells to allow blade outgrowth in the lateral direction. One possible mechanism to maintain cell proliferation is to prevent premature cell differentiation by repressing differentiation-promoting factors at the meristematic domain of the leaf margin. In plants, AS2 is an adaxial cell differentiation-promoting factor.³¹ In stf mutant, the expression of MtAS2 extends from adaxial region to the leaf margin. We found that STF directly binds to several parts of MtAS2 upstream promoter regions to repress MtAS2 expression. Silencing of the MtAS2 expression in stf mutant can partially rescue the blade outgrowth defects,¹⁹ suggesting that AS2 ectopic expression may be part of the reasons for stf mutant narrow leaf phenotype. Interestingly, expression of MtAS2 under the control of the STF promoter in N. sylverstris, resulted in two interesting morphological alterations: Leaf margins are severely up curled to the adaxial side (Fig. 1A and B) and uniform circular knots are formed near the tips of major veins extending from the middle mesophyll to the leaf margin (Fig. 1C). These phenotypic alternations are correlated to STF expression pattern and suggest that AS2 must be repressed in these regions for maintenance of proper cell proliferation. These transgenic plants harboring the STF:MtAS2 construct could be useful for further research to mark the meristematic domain in the leaf.

Figure 1.

Ectopic expression of MtAS2 under STF promoter in N. sylverstris. (A) The adaxial leaf surface of proSTF:MtAS2 compared to WT; (B) The abaxial leaf surface of proSTF:MtAS2 compared to WT; (C) Close view of the abaxial leaf surface of the proSTF:MtAS2 compared to WT. Note that knots are formed between secondary veins and at the end of minor veins.

We previously showed that WUS from Arabidopsis can substitute STF function when expressed under the STF promoter, suggesting that WUS can recognize STF's target(s) in addition to the ability to recruit the co-repressor TPL.^16,22 Indeed, a recent work has revealed that WUS can directly bind and repress differentiation promoting factors, such as KANADI (KAN), AS2 and FILAMENTOUS FLOWER (FIL) in the SAM in Arabidopsis.³² However, the biological significance of the repression of AS2 by WUS for plant development is unclear since AS2 is not expressed in the WUS expression domain of the organization center. To address this, we ectopically expressed AS2 under the control of the WUS promoter³³ in Arabidopsis. Compared with the control plant, the proWUS:AS2 plants showed very narrow and up-curled leaves (Fig. 2A and B). This suggests that either the repression of AS2 by WUS in the SAM is important for leaf blade outgrowth, which is consistent with the importance of a SAM-generated signal in leaf blade development,^34,35 or WUS is expressed at low levels in leaf primordia and may have escaped detection but functions in leaf development redundantly with WOX1 and PRS. Moreover, the inflorescence and flower structures showed phenotypic similarities to the wus mutant, such as aerial rosettes and missing and delayed development of inner organs in the flower (Fig. 2C and D). Thus, ectopic expression of AS2 in WUS's domain strongly affects development of lateral organs, providing evidence that repression of AS2 by WUS has functional significance in plant development.

Figure 2.

Phenotypes of expression of AtAS2 under WUS promoter. (A) A typical 20-day old proWUS:AtAS2 seedling. (B) Compared with Col leaf (left), the proWUS:AtAS2 transgenic plants showed narrow and up-curling leaf. (C) The proWUS:AtAS2 plants showed aerial rosettes phenotype (arrow). (D) The abnormal flower of proWUS:AtAS2 flowers (arrow indicates delayed growth of stamens).

We next examined whether the as2 mutation could rescue the wus mutant phenotype. An as2 loss-of-function mutant (backcrossed to Col) was crossed to the wus mutant (a GABI line). The as2 wus double mutant showed additive phenotypes of wus and as2, such as asymmatric leaves, premature termination of shoot apical meristems and flowers without inner organs (Fig. 3). We also crossed as1 with wus. Like the as2 wus double mutant, the as1 wus double mutant also showed additive phenotypes (Fig. 3). Thus, although AS2 expression is elevated in the wus mutant and ectopic expression of AS2 in WUS domain causes developmental abnormities, the loss-of-function of as2 failed to rescue the wus mutant, suggesting more complex patterns of genetic interactions. One possible explanation for this may be that WUS has many direct targets,³⁶ and repression of AS2 may be only a tip of the iceberg. Alternatively, both cell division and differentiation are required for plant development, and the combination of mutations in both processes gives an even stronger growth defects rather than cross complementation.

Figure 3.

Phenotypes of wus as1 and wus as2 double mutants. The wus as1 and wus as2 double mutants showed additive phenotypes of single mutants.

Mutation of wus Enhances the wox1 prs Mutant Phenotype

In Arabidopsis leaf primordia, WOX1, the STF ortholog, together with PRS function by repressing AS2 at the leaf margin and restricting AS2 expression in the adaxial region.¹⁸ Although WUS expression hasn't been detected in leaves, the wus mutation can result in the development of shoots without juvenile leaves.³⁷ Because AS2 expression under the control of the WUS promoter has a strong leaf phenotype, we wondered whether WUS indeed has a role in blade outgrowth. To address this issue, we crossed the wox1 prs double mutant with the wus mutant and analyzed the phenotypes of the wox1 prs wus triple mutant. The data presented in Fig. 4 shows that the wus mutation enhances the wox1 prs mutant leaf phenotype. The wox1 prs wus triple mutant showed an even narrower leaf phenotype in both the rosette and cauline leaves compared to the wox1prs double mutant (Fig. 4A and B). The leaves of wox1 prs wus triple mutant in juvenile stage were more up-curled. These observations suggest that WUS function is clearly required for Arabidopsis leaf blade development and this may be associated at least in part with the repression of AS2 by WUS. Given that WUS is not expressed in the leaf, it's likely that WUS's effect on leaf development is non-cell autonomous.

Figure 4.

Phenotypes of wox1 prs and wox1 prs wus mutants. (A) The wox1 prs wus triple mutant plants at juvenile stage. (B) The wox1 prs wus triple mutant plant at reproductive stage. Both rosettes and cauline leaves become much narrower than the wox1 prs double mutant (left) grown under same condition.

Taken together, our results suggest that STF and other WUS clade WOX genes involved in stem cell maintenance including the founding member WUS, may have a common mechanism of action: forming a WOX-TPL repressor complex to repress cell differentiation factors and allow cell proliferation. The fact that WUS can target some of leaf polarity patterning genes suggests that there is not only conservation in mechanistic action but also some level of redundancy in targeting the same factors which may have acquired specificity through evolution to allow the development of a complex architecture independently controlled at different tissue levels. It is interesting to note that highly conserved and ubiquitously expressed TPL co-repressors are recruited by multiple plant-specific and tissue specific transcription factors that carry out crucial functions in specific tissues. Especially, the WOX-TPL interaction highlights nature's conservative model since WOX genes are involved in a wide array developmental programs throughout the plant life cycle. How the WOX-TPL complex achieves transcriptional repression is an exciting question to address with further research, and the leaf blade may provide a convenient genetic tool to dissect the mechanistic molecular details with direct assessment of phenotypic consequences.

Funding

This work was supported by the National Science Foundation Grant IOS-1354422.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Drs. Michiel Vandenbussche for providing wox1 prs seeds and Jennifer Fletcher for providing the as2-1 seeds. We also thank TAIR for providing the as1-1 and wus-gabi seeds.