Sharyn E. Perry

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.

Global Identification of Targets of the Arabidopsis MADS Domain Protein AGAMOUS-Like15

AGAMOUS-Like15 (AGL15) is a MADS domain transcriptional regulator that promotes somatic embryogenesis by binding DNA and regulating gene expression. Chromatin immunoprecipitation (ChIP) analysis previously identified DNA fragments with which AGL15 associates in vivo, and a low-throughput approach revealed a role for AGL15 in gibberellic acid catabolism that is relevant to embryogenesis. However, higher throughput methods are needed to identify targets of AGL15. Here, we mapped AGL15 in vivo binding sites using a ChIP-chip approach and the Affymetrix tiling arrays for Arabidopsis thaliana and found that ∼2000 sites represented in three biological replicates of the experiment are annotated to nearby genes. These results were combined with high-throughput measurement of gene expression in response to AGL15 accumulation to discriminate responsive direct targets from those further downstream in the network. LEAFY COTYLEDON2, FUSCA3, and ABA INSENSITIVE3, which encode B3 domain transcription factors that are key regulators of embryogenesis, were identified and verified as direct target genes of AGL15. Genes identified as targets of the B3 genes are also targets of AGL15, and we found that INDOLEACETIC ACID-INDUCED PROTEIN30 is involved in promotion of somatic embryo development. The data presented here and elsewhere suggest that much cross-regulation occurs in gene regulatory networks underpinning embryogenesis.

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.

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.

Expression and Maintenance of Embryogenic Potential Is Enhanced through Constitutive Expression of AGAMOUS-Like 15

The MADS domain protein AGL15 (AGAMOUS-Like 15) has been found to preferentially accumulate in angiosperm tissues derived from double fertilization (i.e. the embryo, suspensor, and endosperm) and in apomictic, somatic, and microspore embryos. Localization to the nuclei supports a role in gene regulation during this phase of the life cycle. To test whether AGL15 is involved in the promotion and maintenance of embryo identity, the embryogenic potential of transgenic plants that constitutively express AGL15 was assessed. Expression of AGL15 was found to enhance production of secondary embryos from cultured zygotic embryos, and constitutive expression led to long-term maintenance of development in this mode. Ectopic accumulation of AGL15 also promoted somatic embryo formation after germination from the shoot apical meristem of seedlings in culture. These results indicate that AGL15 is involved in support of development in an embryonic mode.

The Embryo MADS Domain Protein AGAMOUS-Like 15 Directly Regulates Expression of a Gene Encoding an Enzyme Involved in Gibberellin Metabolism

AGL15 (for AGAMOUS-Like 15) is a member of the MADS domain family of DNA binding transcriptional regulators that accumulates to its highest amounts during embryo development. To better understand how AGL15 functions, a chromatin immunoprecipitation approach was used to identify directly regulated genes. One DNA fragment that coprecipitated with AGL15 corresponded to a portion of the regulatory region of a gene named DTA1 (for Downstream Target of AGL15-1). The expression of DTA1 was positively correlated with AGL15 abundance during embryogenesis. In this report, a cis element for response to AGL15 was identified, and the activity of DTA1 as a gibberellin (GA) 2-oxidase was confirmed. DTA1 corresponds to AtGA2ox6 and was renamed to indicate this identity. Further experiments related the function of AtGA2ox6 to regulation by AGL15. Constitutive expression of AGL15 and of AtGA2ox6 altered endogenous GA amounts and caused GA-deficient phenotypes in Arabidopsis thaliana that could be at least partially rescued by application of biologically active GA. The phenotype of plants with decreased expression of AtGA2ox6 was the converse of plants overexpressing AtGA2ox6 in terms of seed germination attributes and effects on somatic embryo production.

Identification of Direct Targets of FUSCA3, a Key Regulator of Arabidopsis Seed Development

A set of genes expressed during Arabidopsis embryo development are directly controlled by the transcription factor FUSCA3. FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box-binding factor. LEC1, LEC2, and FUS3 are essential for plant embryo development. All three loss-of-function mutants in Arabidopsis (Arabidopsis thaliana) prematurely exit embryogenesis and enter seedling developmental programs. When ectopically expressed, these genes promote embryo programs in seedlings. We report on chromatin immunoprecipitation-tiling array experiments to globally map binding sites for FUS3 that, along with other published work to assess transcriptomes in response to FUS3, allow us to determine direct from indirect targets. Many transcription factors associated with embryogenesis are direct targets of FUS3, as are genes involved in the seed maturation program. FUS3 regulates genes encoding microRNAs that, in turn, control transcripts encoding transcription factors involved in developmental phase changes. Examination of direct targets of FUS3 reveals that FUS3 acts primarily or exclusively as a transcriptional activator. Regulation of microRNA-encoding genes is one mechanism by which FUS3 may repress indirect target genes. FUS3 also directly up-regulates VP1/ABI3-LIKE1 (VAL1), encoding a B3 domain protein that functions as a repressor of transcription. VAL1, along with VAL2 and VAL3, is involved in the transition from embryo to seedling development. Many genes are responsive to FUS3 and to VAL1/VAL2 but with opposite regulatory consequences. The emerging picture is one of complex cross talk and interactions among embryo transcription factors and their target genes.

The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development.

Little is known about regulatory factors that act during the earliest stages of plant embryogenesis. The MADS domain protein AGL15 (for AGAMOUS-like) is expressed preferentially during embryogenesis and accumulates during early seed development in monocotyledonous and dicotyledonous flowering plants. AGL15-specific antibodies and immunohistochemistry were used to demonstrate that AGL15 accumulates before fertilization in the cytoplasm in the cells of the egg apparatus and moves into the nucleus during early stages of development in the suspensor, embryo, and endosperms. Relatively high levels of AGL15 are present in the nuclei during embryo morphogenesis and until the seeds start to dry in Brassica, maize, and Arabidopsis. AGL15 is associated with the chromosomes during mitosis, and gel mobility shift assays were used to demonstrate that AGL15 binds DNA in a sequence-specific manner. To assess whether AGL15 is likely to play a role in specifying the seed or embryonic phase of development, AGL15 accumulation was examined in Arabidopsis mutants that prematurely exit embryogenesis. lec1-2 mutants show an embryo-specific loss of AGL15 at the transition stage, suggesting that AGL15 interacts with regulators in the leafy cotyledons pathway.

In vitro import of proteins into chloroplasts

Reconstitution of precursor transport using isolated intact chloroplasts was first demonstrated in the late 1970s using the precursor to the small subunit of ribulose bis-phosphate carboxylase [1,2]. Subsequent optimization and characterization has demonstrated that the in vitro reconstitution assay works with a number of precursor proteins [3] and faithfully mimics intra-organellar targeting as well as transport into the chloroplast [4, 5]. The in vitro reconstitution assays have been very useful in studying the details of the transport process as well as confirming the intracellular location of putative chloroplastic precursor proteins after the isolation of cDNA clones [6, 5]. Those readers desiring a detailed study of precursor protein transport into chloroplasts should consult the extensive literature on this topic [7–9]. This chapter is aimed at researchers with a cDNA clone encoding a putative precursor protein who wish to evaluate the transport competence of the precursor to confirm the intracellular location of the gene product.

The MADS-Domain Transcriptional Regulator AGAMOUS-LIKE15 Promotes Somatic Embryo Development in Arabidopsis and Soybean

The MADS-domain transcriptional regulator AGAMOUS-LIKE15 (AGL15) has been reported to enhance somatic embryo development when constitutively expressed. Here we report that loss-of-function mutants of AGL15, alone or when combined with a loss-of-function mutant of a closely related family member, AGL18, show decreased ability to produce somatic embryos. If constitutive expression of orthologs of AGL15 is able to enhance somatic embryo development in other species, thereby facilitating recovery of transgenic plants, then AGL15 may provide a valuable tool for crop improvement. To test this idea in soybean (Glycine max), a full-length cDNA encoding a putative ortholog of AGL15 was isolated from soybean somatic embryos. Subsequently, the corresponding genomic region of the gene was obtained. This gene, designated GmAGL15, encodes a protein with highest similarity to AGL15 from Arabidopsis (Arabidopsis thaliana) and Brassica napus that accumulates to its highest amount in embryos in these species. Like Arabidopsis and Brassica AGL15, GmAGL15 was preferentially expressed in developing embryos. When ectopically overexpressed the soybean protein was able to enhance somatic embryo development in soybean.