The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.     

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.     

Endosperm cellularization defines an important developmental transition for embryo development

The endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endosperm develops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occurs at a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidy crosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seed development. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) and endosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endosperm cellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryo development, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endosperm cellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is a consequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct target gene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endosperm cellularization.     

Control of expression and autoregulation of AGL15, a member of the MADS-box family

AGL15 is an Arabidopsis thaliana MADS-domain regulatory factor that not only preferentially accumulates during embryogenesis but is also expressed at lower levels after the completion of germination. To better understand the control of expression of AGL15, a series of 5' and internal deletions within the regulatory regions of AGL15 was generated. Regions important for the level of expression, including a region involved in expression in response to auxin, were identified. Additionally, AGL15 expression was found to respond to AGL15 accumulation amounts and to altered forms of AGL15. This feedback loop is at least in part due to direct regulation, as assessed by in vivo and in vitro binding of AGL15 to its own regulatory regions and by site-directed mutagenesis studies.     

The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression

AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunochemistry, we found that the AGL15 protein accumulates transiently in the shoot apex in young Arabidopsis and Brassica seedlings and that promoter activity is associated with the shoot apex and the base of leaf petioles throughout the vegetative phase. During the reproductive phase, AGL15 accumulates transiently in floral buds. When AGL15 was expressed in Arabidopsis under the control of a strong constitutive promoter, we noted a striking increase in the longevity of the sepals and petals as well as delays in a selected set of age-dependent developmental processes, including the transition to flowering and fruit maturation. Although ethylene has been implicated in many of these same processes, the effects of AGL15 could be clearly distinguished from the effects of the ethylene resistant1-1 mutation, which confers dominant insensitivity to ethylene. By comparing the petal breakstrength (the force needed to remove petals) for flowers of different ages, we determined that ectopic AGL15 had a novel effect: the breakstrength of petals initially declined, as occurs in the wild type, but was then maintained at an intermediate value over a prolonged period. Abscission-associated gene expression and structural changes were also altered in the presence of ectopic AGL15.     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

Soybean Seed Protein Genes Are Regulated Spatially during Embryogenesis

We used in situ hybridization to investigate Kunitz trypsin inhibitor gene expression programs at the cell level in soybean embryos and in transformed tobacco seeds. The major Kunitz trypsin inhibitor mRNA, designated as KTi3, is first detectable in a specific globular stage embryo region, and then becomes localized within the axis of heart, cotyledon, and maturation stage embryos. By contrast, a related Kunitz trypsin inhibitor mRNA class, designated as KTi1/2, is not detectable during early embryogenesis. Nor is the KTi1/2 mRNA detectable in the axis at later developmental stages. Outer perimeter cells of each cotyledon accumulate both KTi1/2 and KTi3 mRNAs early in maturation. These mRNAs accumulate progressively from the outside to inside of each cotyledon in a "wave-like" pattern as embryogenesis proceeds. A similar KTi3 mRNA localization pattern is observed in soybean somatic embryos and in transformed tobacco seeds. An unrelated mRNA, encoding [beta]-conglycinin storage protein, also accumulates in a wave-like pattern during soybean embryogenesis. Our results indicate that cell-specific differences in seed protein gene expression programs are established early in development, and that seed protein mRNAs accumulate in a precise cellular pattern during seed maturation. We also show that seed protein gene expression patterns are conserved at the cell level in embryos of distantly related plants, and that these patterns are established in the absence of non-embryonic tissues.     

The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis

The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18.     

The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.     

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.     

AtBUD13 affects pre‐mRNA splicing and is essential for embryo development in Arabidopsis

Pre‐mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud‐site selection protein 13) regulates the budding pattern and pre‐mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T‐DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA‐seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre‐mRNA splicing.