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The seed is a key evolutionary adaptation of land plants that facilitates dispersal and allows for germination when the environmental
conditions are adequate. Mature seeds are dormant and desiccated, with accumulated storage products that are to be used by
the seedling after germination. These properties are imposed on the developing embryo by a maturation program, which operates
during the later part of embryogenesis. A number of “master regulators” (the “LEC genes”) required for the induction of the
maturation program have been described, but it is not known what prevents this program from being expressed during early embryogenesis.
Here, we report that Arabidopsis (Arabidopsis thaliana) embryos mutant for strong alleles of DICER-LIKE1, the enzyme responsible for the biosynthesis of microRNAs (miRNAs), mature earlier than their wild-type counterparts. This
heterochronic phenotype indicates that miRNAs are key regulators of the timing of the maturation program. We demonstrate that
miRNAs operate in part by repressing the master regulators LEAFY COTYLEDON2 and FUSCA3 and identify the trihelix transcription factors ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1) and ASIL2 and the histone deacetylase HDA6/SIL1 as components that act downstream of miRNAs to repress the maturation program early in embryogenesis. Both ASIL1 and HDA6/SIL1 are known to act to prevent the expression of embryonic maturation genes after germination, but to our knowledge, this is
the first time they have been shown to have a role during embryogenesis. Our data point to a common negative regulatory module
of maturation during early embryogenesis and seedling development.
One of the reasons for the evolutionary success of seed plants is their ability to generate a resistant structure, the seed,
which facilitates dispersal and reinitiates development only in the appropriate environmental conditions. In Arabidopsis (Arabidopsis thaliana), wild-type embryos follow a predictable pattern of cell divisions, going through a series of stages named after the shape
of the embryo: preglobular, globular, transition, heart, torpedo, bent green cotyledon, and mature (Jürgens and Mayer, 1994). These stages encompass two major phases of development. The first part of embryogenesis, until the heart stage, is devoted
to patterning, setting up the embryonic axes, meristems, and tissue types (Jenik et al., 2007). The heart-to-late-heart stage transition marks the onset of embryonic maturation, first evidenced by the appearance of
chlorophyll autofluorescence in the epidermis of the hypocotyl, signaling the beginning of proplastid maturation to chloroplasts
(Mansfield and Briarty, 1991). The embryos turn green in color and start accumulating storage products at the early torpedo stage. The photosynthetic
activity of the embryonic chloroplasts may contribute to the biosynthesis of storage lipids (Ruuska et al., 2004). In Arabidopsis, the storage products include seed storage proteins and storage lipids (very-long-chain fatty acids, polyunsaturated
fatty acids, and triacylglycerols; Baud et al., 2008). Once the embryos fill the seed, they acquire desiccation tolerance, desiccate, and enter dormancy (Vicente-Carbajosa and Carbonero, 2005; Baud et al., 2008).
Because the maturation program directs seed dormancy, it needs to be carefully timed, ensuring it starts midembryogenesis
and is repressed after germination. A complex network is involved in timing maturation during embryo development, including
positive regulators of maturation during midembryogenesis and negative regulators after germination. The existence of repressors
of maturation in early embryogenesis has been postulated, but none have been identified so far (for review, see Baud et al., 2008; Santos-Mendoza et al., 2008).
The central positive regulators of the seed maturation program are the “LEC genes” (the B3 domain transcription factors LEAFY-COTYLEDON2
[LEC2] and FUSCA3 [FUS3] and the B subunits of the NF-Y family of trimeric transcription factors LEC1 [also called NF-YB9]
and LEC1-LIKE [L1L, NF-YB6]) and the B3 domain transcription factor ABA INSENSITIVE3 (ABI3). Single loss-of-function mutations
in all of these genes result in deficiencies in the accumulation of storage products, desiccation intolerance, and/or a heterochronic
change of cotyledons into true leaves. These factors regulate each other at the transcriptional level in embryos, and their
interactions vary depending on tissue type. The hormones abscisic acid (ABA) and GA interact with this network of transcription
factors. High ratios of ABA to GA promote maturation via ABI3 and ABI5 (Santos-Mendoza et al., 2008). In vitro studies have suggested that LEC1 and L1L interact with NY-YC1, -2, and -6 to up-regulate the induction of storage
product genes by ABA (Yamamoto et al., 2009). Several lines of evidence have identified other positive regulators, acting either at the same level or downstream of the
LEC genes, by binding to the promoters of genes encoding storage proteins and other genes involved in seed maturation: bZIP
transcription factors that cooperate with the NF-Y complexes (ABI5, bZIP10, bZIP25, bZIP53, and bZIP67; Bensmihen et al., 2002; Alonso et al., 2009; Yamamoto et al., 2009); MYB transcription factors (AtMYB115 and AtMYB118; Zhang et al., 2009; Wang et al., 2009); and MADS box transcription factors (AGAMOUS-LIKE15 [AGL15] and AGL18; Zheng et al., 2009).
A number of factors are known to prevent the expression of embryonic traits after seed germination (for review, see Zhang and Ogas, 2009). This negative regulation involves transcriptional mechanisms via the B3 domain proteins VP1/ABI3-LIKE or HIGH-LEVEL EXPRESSION
OF SUGAR-INDUCIBLE GENE (VAL1/HSI2) and VAL2/HSL1 and the trihelix protein ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1).
Epigenetic factors also repress the maturation program in seedlings, including histone deacetylases (HDA6/SIL1 and HDA19),
Polycomb group proteins (SWINGER, CURLY LEAF, and MEDEA), and chromatin remodelers (BRAHMA, AtSWI3c, BSH, and PICKLE). These
transcriptional and epigenetic regulators appear to act both directly and indirectly (by modulating the actions of GA) to
prevent the expression of the LEC genes and of the genes encoding storage products. It is not known whether any of these regulators
or other factors are responsible for the repression of the maturation early in embryogenesis.
MicroRNAs (miRNAs) are 21-nucleotide single-stranded RNA molecules that act by binding complementary target mRNAs to promote
their cleavage or interfere with translation (for review, see Voinnet, 2009). miRNAs are generated by cleavage of a precursor miRNA by a complex that includes the RNase III DICER-LIKE1 (DCL1), the
double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2-zinc finger protein SERRATE (SE). The resulting
miRNAs are then methylated by HUA-ENHANCER1 (HEN1) and incorporated into an RNA-induced silencing complex, which leads the
miRNA to its target and effects either cleavage or repression of translation. ARGONAUTE proteins such as AGO1 and AGO10/ZWILLE/PINHEAD
are central components of miRNA RNA-induced silencing complexes. Null alleles of dcl1 are embryonic lethal, but embryos mutant for other elements of the miRNA pathway have either nonoverlapping phenotypes or
no observable phenotype (Lynn et al., 1999; Lu and Fedoroff, 2000; Chen et al., 2002; Grigg et al., 2005; Ronemus et al., 2006; Kurihara et al., 2009).
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