Control of dicot leaf blade expansion by a WOX gene, STF

Abstract

WUSCHEL-RELATED HOMEOBOX (WOX) genes are plant specific transcription factors that serve as master switches controlling key developmental programs from embryo apical-basal asymmetric patterning to organizing stem cells and development of lateral organs. Recently, we reported the requirement of a WOX1/MAW-like gene, STENOFOLIA (STF), for blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris. The stf mutant in Medicago produces narrow leaves where mediolateral outgrowth of the blade is severely curtailed while proximodistal growth and trifoliate identity remain unaffected. The lam1 mutant in N. sylvestris produces leaves devoid of blade tissue with just 1-2 layers of rudimentary strips and lacks stem elongation. stf and lam1 mutants have narrow petals and are female sterile due to defective ovule development. Morphological analysis of mutants and STF expression patterns suggest that STF regulates blade outgrowth mainly by controlling cell division in the margins of leaf primordium. Both the blade and flower phenotypes of lam1 can be complemented with WUS expressed under the STF promoter suggesting a conserved mechanism in stem cell maintenance and lateral organ development.

In dicotyledonous plants, leaf primordia are initiated at regular intervals at the flanks of the shoot apical meristem (SAM) where actively dividing cells are recruited from the peripheral zone of the SAM and serve as leaf founder initials.¹ Leaf morphogenesis requires cell division and cell expansion of the initial founder cells supplemented with additional recruitment of cell files from the SAM to establish an expanded lamina differentiated in three axises; proximodistal, mediolateral and dorsoventral. Although dorsoventral differentiation is thought to be a prerequisit for lamina outgrowth,² the presence of mutants in Arabidopsis that are affected only in proximodistal or mediolateral growth suggested that different mechanisms of cell proliferation and expansion patterns may exist to accomplish growth in length and width directions.³

STF is required for cell division in marginal meristems

The stf and lam1 mutants are severely affected only in mediolateral (width) growth of the leaf blade and flower petal where both cell division and cell expansion are compromized. For example, mutant blade cells are expanded to approximately 75% of wild-type, and there are at least 3-fold fewer cells in stf blade in the mediolateral axis along the medial region suggesting that cell division accounts for most of the defects in blade expansion.⁴ This could be, at least in theory, due to recruitment of fewer blade initial cells from the SAM into the leaf primordium, slower rate of cell division in the meristematic ground tissue of the leaf, or premature exit from the mitotic cell cycle. The STF related WOX gene in Arabidopsis, PRS1/WOX3,^5,6 or NS1 and NS2 in maize^6-8 are required for recruitment of founder initials into the lateral organs primordia, and the wox1 prs1 or ns1 ns2 double mutants produce narrow leaves in Arabidopsis and maize, respectively.^8,9 However, recruitment of blade founder initials from the SAM is less likely to be the function of STF/LAM1 for the following reasons. First, in lam1 leaf primordium, blade founder initials are recruited normally in a manner comparable to wild-type but organized lamina outgrowth fails.¹⁰ Second, in stf leaf primordium, leaf founders are recruited at the right position and blade tissue is formed in both the terminal leaflet and the two lateral leaflets, but expansion of the lamina and further differentiation of tissues is arrested leaving the width of stf leaves less than a third of the wild-type.⁴ Besides, the STF gene is expressed at the adaxial-abaxial junction of the leaf margin throughout leaf growth and disappears in the mature fully expanded leaf suggesting that STF function is an ongoing requirement until blade expansion ceases. Moreover, sgl1and palm1 mutants in Medicago having unifoliate and pentafoliate leaves, respectively,^11,12 instead of the normal trifoliate which are likely to have defects associated with recruitment of blade founders, appear to have normal mediolateral growth suggesting that blade specification and elaboration are not necessarily controlled by the same genetic factors. Similarly, the Medicago slm1 mutant is affected in lateral and terminal leaflet number without significant defect in lamina expansion.¹³ Third, both stf and lam1 leaves are indistinguishable from wild-type in proximodistal growth except that the vasculature including the midrib is poorly developed.⁴ There is no evidence to suggest that two different groups of blade founder cells, one group for proximodistal growth and the second group for lateral growth, are recruited into the leaf promordium. Rather, a group of uniform leaf founder initial cells are recruited from the SAM, and tightly regulated cell division patterns followed by cell expansion (enlargement) at specific developmental stages could establish growth in both the length and width directions. It is, therefore, likely that the stf and lam1 defects are associated with cell division although this could take the form of slower cell divion rate or premature exit from the cell cycle in the leaf margin. We propose that STF/LAM1 regulates cell division in the marginal meristem (blastozone) by directly or indirectly controlling the activity of GRAS genes, specific cyclins and their cognate protein kinases. Consistent with this hypothesis, KNOX1, LOB, BTB/POZ domain genes have been upregulated, and SHR-related GRAS genes, hormone responsive MYB genes and D-type cyclins have been significantly downregulated in the stf mutant suggesting that coordinating a balance between repression and activation functions is required for proper lamina outgrowth. The cell expansion defect of stf and lam1 mutants may be dependent on cell division derived signals and could be a consequence of the cell division defects as cell division and cell expansion events are closely linked during leaf growth (M. Andriankaja personal communication).

STF is specific to dicots

The STF protein is homologous to Arabidopsis WOX1 and petunia MAW, and is closely related to PFS2/WOX6, WUS and PRS1/WOX3. Phylogenetic analysis based on full length amino acid sequences or conserved motifs revealed that WOX1/MAW/STF from different species form one subclade separate from WUS and WOX3 subclades.^4,9,14,15 STF is represented by a small gene family having one or two members in all the sequenced genomes of dicotyledenous species, but we were unable to identify obvious STF homologs in the genomes of rice, maize, sorghum, false brume, foxtail millet and other monocotyledenous ESTs including wheat, barley and banana (Fig. 1). This suggests that STF may be acquired for extended lamina flattening and network venation patterns after dicots and monocots are separated. This is not surprising given the evolutionarily dynamism of the WOX gene family.^15-17In Arabidopsis, the WOX family comprises 15 members including WUS¹⁸ and has been divided into three major clades. The ancient or basal clade represented by WOX10, 13 and 14 is detected in as early as green algae and mosses; the intermediate clade represented by WOX8, 9,11 and 12 appeared in vascular plants including lycophytes; and the modern clade also called the WUS clade represented by WUS and WOX1–7 is found only in seed plants.^15-17 Even among the modern clade, WUS and WOX5 are represented by only one ancestral sequence in gymnosperms but by two distinct, though functionally interchangeable, sequences in angiosperms.¹⁵ For this reason, WOX5 has been considered as the evolutionary innovation of angiosperms marking the 300–350 million years of evolution where the angiosperms and gymnosperms separate.¹⁵ It is conceivable that STF/WOX1 points to yet another important land mark of aproximately 140 million years of independent evolution separating dicots from monocots.

Figure 1.

Phylogenetic analysis of WUS, PRS and WOX1 like proteins within the modern clade of WOX class transcription factors. The phylogenetic tree was constructed using 72 amino acids around the homeodomain region and the MEGA4 software. Bootstrap values were generated from 1000 trials. Note that the WUS, PRS and STF/MAW like proteins from different species form three distinct subclades statistically well supported by bootstrap values. Both the WUS and PRS subclades are represented by members from dicots and monocots in which the monocot and dicot sequences of each subclade group separately within their respective subclade. Monocot sequences representing the STF/MAW subclade have not been identified in the sequenced genomes and extensive EST databases available to date.

Tadege M, Lin H, Bedair M, Berbel A, Wen J, Rojas CM, et al. STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris. Plant Cell. 2011;23:2125–42. doi: 10.1105/tpc.111.085340.