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Early dcl1 mutant embryos lack miR156 and exhibit premature expression of miR156 targets SPL10 and SPL11, which in turn induces precocious expression of genes normally induced during the maturation phase of embryogenesis. We propose
that additional plant miRNAs—including miR160, miR166, and miR319—also forestall expression of differentiation-promoting transcription
factors such as ARF17, CNA, PHB, PHV, and TCP4. A link between delayed reduction of maternal transcripts and the morphogenesis phenotypes observed in MZdicer embryos awaits
experimental confirmation. The same is true for the precocious expression of maturation transcripts and the patterning phenotypes
observed in dcl1 embryos, although SPL10 and SPL11 have been experimentally linked to defects in both patterning and maturation gene expression.
One of the earliest perceptible roles of plant miRNAs is to repress transcription factors (Fig. 2C). We propose that, by repressing differentiation-promoting transcription factors (including SPL10, SPL11, ARF17, CNA, PHB, PHV, and TCP4), miRNAs maintain the potential of preglobular cells to generate diverse cell types at the subsequent globular stages. Since
the reduction of WOX2 transcripts in dcl1 eight-cell embryos was the first detectable differentiation defect, and homeobox genes related to WOX2 have been implicated in maintaining stem cells during post-embryonic development (Laux et al. 1996; Mayer et al. 1998; Wu et al. 2005), we speculate that premature expression of miRNA targets leads to the reduction of WOX2 transcripts in dcl1 embryos and ultimately contributes to precocious differentiation and subsequent loss of developmental potential in preglobular
cell types.
The two transcription factor genes that were most derepressed in eight-cell dcl1 embryos were miR156 targets SPL10 and SPL11, for which transcripts were up-regulated ≥150-fold (≥600-fold if only the homozygous mutant embryos contributed to the increase).
These two genes were redundantly required for the embryonic patterning defects observed in dcl1 embryos (Table 1; Fig. 4A,D). The ability to suppress such a pleiotropic phenotype by knocking out/down only two miRNA targets was surprising when considering
that dozens of miRNA targets were derepressed in dcl1 embryos. However, miR156-resistant SPL10 and SPL11 transgenes did not phenocopy dcl1 embryos, which suggests that the misregulation of additional targets contributes to the patterning defects observed. Consistent
with this idea, at least seven miRNA target transcripts in addition to SPL10 and SPL11 have increased levels as early as the eight-cell stage, and all of these encode transcription factors.
Of these seven misregulated targets, five (ARF17, CNA, PHB, PHV, and TCP4) have reported embryonic functions after the preglobular stage (Palatnik et al. 2003; Mallory et al. 2005; Prigge et al. 2005). miR166-mediated regulation of PHB and PHV has also been reported to be important during early embryonic patterning (Grigg et al. 2009). However, phb phv double mutants do not suppress dcl1-null embryonic phenotypes (Grigg et al. 2009), suggesting that the misregulation of other miRNA targets is required for the dcl1 embryonic phenotypes. Perhaps the loss of both miR156-mediated regulation of SPL transcripts and miR166-mediated regulation
of HD-ZIPIII transcripts in dcl1 embryos is responsible for the embryonic patterning phenotypes observed. Future characterization of how multiple miRNA/target
interactions facilitate embryonic cell differentiation will contribute to a better understanding of how the basic plant body
plan is established during embryo morphogenesis.
We also report previously uncharacterized roles of miR156-mediated SPL gene repression during embryogenesis. After morphogenesis, seed plant embryos transition to a maturation phase, when they
accumulate storage proteins, undergo desiccation tolerance, and prepare to enter into a state of dormancy prior to germination.
We found that miR156-mediated regulation of SPL10 and SPL11 prevents the transcription factor products of these genes from prematurely inducing seed maturation genes before the embryo
has formed. To the extent that this regulation forestalls the morphogenesis-to-maturation-phase transition, this newly identified
role for miR156-mediated repression of SPL genes resembles that observed for vegetative-, reproductive-, and meristem identity-phase transitions during post-embryonic
development (Wu and Poethig 2006; Gandikota et al. 2007; Schwarz et al. 2008; Wang et al. 2009; Wu et al. 2009; Yamaguchi et al. 2009). This role for SPL10 and SPL11 repression might be tied to the requirement of these targets for the patterning defects observed in dcl1 embryos. Perhaps the premature induction of maturation-phase genes arrests morphogenesis before it is complete.
Later in development, the miR156-mediated repression of SPL10 and SPL11 must presumably be overcome in order for the embryo to undergo the morphogenesis-to-maturation transition. Although miR156
levels do not appear to decrease at later stages of morphogenesis (Fig. 3A), SPL10 and SPL11 promoter activities do increase at the same stages that the morphogenesis-to-maturation-phase transition occurs (Fig. 5). Therefore, at later stages of morphogenesis, miR156 may establish a threshold that SPL10 and SPL11 transcript levels must surpass in order to accumulate and promote maturation-phase gene expression programs. Despite the
characterization of hormonal, metabolic, and genetic factors involved in embryo maturation (Nambara and Marion-Poll 2003; Weber et al. 2005; Braybrook and Harada 2008), the regulatory mechanisms that control the morphogenesis-to-maturation-phase transition are still not well understood.
Multiple factors may influence SPL10 and SPL11 transcript levels and ultimately regulate the timing of the morphogenesis-to-maturation-phase transition. Further characterization
of the embryonic functions of SPL transcription factors may provide a handle to acquire a better molecular understanding of the morphogenesis-to-maturation-phase
transition.
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