A homolog of GuidedEntry of Tail-anchored proteins3 functions in membrane-specific protein targeting in chloroplasts of Arabidopsis.

The chloroplasts and mitochondria of photosynthetic eukaryotes contain proteins that are closely related to cytosolic Guided Entry of Tail-anchored proteins3 (Get3). Get3 is a targeting factor that efficiently escorts tail-anchored (TA) proteins to the ER. Because other components of the cytosolic-targeting pathway appear to be absent in organelles, previous investigators have asserted that organellar Get3 homologs are unlikely to act as targeting factors. However, we show here both that the Arabidopsis thaliana chloroplast homolog designated as GET3B is structurally similar to cytosolic Get3 proteins and that it selectively binds a thylakoid-localized TA protein. Based on genetic interactions between a get3b mutation and mutations affecting the chloroplast signal recognition particle-targeting pathway, as well as changes in the abundance of photosynthesis-related proteins in mutant plants, we propose that GET3B acts primarily to direct proteins to the thylakoids. Furthermore, through molecular complementation experiments, we show that function of GET3B depends on its ability to hydrolyze ATP, and this is consistent with action as a targeting factor. We propose that GET3B and related organellar Get3 homologs play a role that is analogous to that of cytosolic Get3 proteins, and that GET3B acts as a targeting factor in the chloroplast stroma to deliver TA proteins in a membrane-specific manner.

Membrane-Specific Targeting of Tail-Anchored Proteins SECE1 and SECE2 Within Chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately targeted in order to maintain the identity and function of the highly differentiated internal membranes. Relatively little is known about the targeting information or pathways that direct proteins with transmembrane domains to either the inner envelope or thylakoids. In this study, we focused on a structurally simple class of membrane proteins, the tail-anchored proteins, which have stroma-exposed amino-terminal domains and a single transmembrane domain within 30 amino acids of the carboxy-terminus. SECE1 and SECE2 are essential tail-anchored proteins that function as components of the dual SEC translocases in chloroplasts. SECE1 localizes to the thylakoids, while SECE2 localizes to the inner envelope. We have used transient expression in Arabidopsis leaf protoplasts and confocal microscopy in combination with a domain-swapping strategy to identify regions that contain important targeting determinants. We show that membrane-specific targeting depends on features of the transmembrane domains and the short C-terminal tails. We probed the contributions of these regions to targeting processes further through site-directed mutagenesis. We show that thylakoid targeting still occurs when changes are made to the tail of SECE1, but changing residues in the tail of SECE2 abolishes inner envelope targeting. Finally, we discuss possible parallels between sorting of tail-anchored proteins in the stroma and in the cytosol.

Two paths diverged in the stroma: targeting to dual SEC translocase systems in chloroplasts.

Chloroplasts inherited systems and strategies for protein targeting, translocation, and integration from their cyanobacterial ancestor. Unlike cyanobacteria however, chloroplasts in green algae and plants contain two distinct SEC translocase/integrase systems: the SEC1 system in the thylakoid membrane and the SEC2 system in the inner envelope membrane. This review summarizes the mode of action of SEC translocases, identification of components of the SEC2 system, evolutionary history of SCY and SECA genes, and previous work on the co- and post-translational targeting of lumenal and thylakoid membrane proteins to the SEC1 system. Recent work identifying substrates for the SEC2 system and potential features that may contribute to inner envelope targeting are also discussed.

Sorting of SEC translocase SCY components to different membranes in chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase.

Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40.

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1.

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component.

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid-based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co-immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma-exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase.

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.

Plastids contain a second sec translocase system with essential functions.

Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane.

MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis.

MADS box genes encode transcription factors that play important regulatory roles at various stages in plant development. Transcripts encoding the MIKC*-type (for MADS DNA-binding domain, Intervening domain, Keratin-like domain, and C-terminal domain) factors, a divergent clade, are enriched in mature pollen. Previous studies have shown that these proteins bind DNA as heterodimers, which form between S- and P-class MIKC* proteins. In this study, Arabidopsis (Arabidopsis thaliana) pollen with little or no MIKC* activity was produced by combining strong loss-of-function alleles of the S-class proteins AGAMOUS-LIKE66 (AGL66) and AGL104. Double mutant plants produce pollen but have severely reduced fertility due to reduced pollen viability, delayed germination, and aberrant pollen tube growth. Microarray analysis of the mutant pollen revealed that the loss of MIKC* regulation has a major impact on pollen gene expression. Pollen competition assays involving various combinations of AGL65, AGL66, AGL104, and AGL94 mutant alleles provided genetic evidence that at least three heterodimers (AGL30-AGL104, AGL65-AGL104, and AGL30-AGL66) form and function in at least a partially redundant fashion in pollen. Analyses of transcript abundance in wild-type and mutant pollen indicated that AGL65-containing complexes are likely to be more abundant than the others and that accumulation of AGL30 and AGL94 transcripts increases in response to reductions in MIKC* activity. These results were combined to create a model to describe MIKC* heterodimer contributions in pollen.

Localization and integration of thylakoid protein translocase subunit cpTatC.

Thylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.

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.

Expression of MADS-box genes during the embryonic phase in Arabidopsis.

MADS domain factors play important roles as developmental regulators in plants. In Arabidopsis thaliana, MADS domain proteins have been shown to regulate various processes during the vegetative and reproductive phases. Relatively little is known, however, about family members expressed during the embryonic phase and their function. To determine which MADS-box genes are expressed during the embryonic phase in Arabidopsis, a family-wide survey involving gene-specific primers and RT-PCR was conducted. Transcripts corresponding to 64 (out of 109 total) family members could be detected in RNA samples isolated from embryonic culture tissue. Eight MADS-box genes that appear to be expressed at higher levels during the embryonic phase than in seedlings or in inflorescence apices were identified. The spatial pattern of expression in developing seeds was characterized for four MADS-box genes (FLOWERING LOCUS C, FLOWERING LOCUS M, AGAMOUS-LIKE 15, and AGAMOUS-LIKE 18) using reporter constructs encoding translational fusions to GUS. All four are expressed in cells throughout the endosperm and embryo. Finally, to test the hypothesis that AGAMOUS-LIKE15 (AGL15) and AGAMOUS-LIKE18 (AGL18) play essential roles during the embryonic phase, plants carrying T-DNA insertions that disrupt these genes were isolated. No embryo defects were observed in agl15 or agl18 single mutants or in agl15agl18 double mutants. These results indicate that multiple regulatory pathways that involve MADS domain factors are likely to operate in embryonic tissues, and that genetic and/or functional redundancy are likely to be as prevalent as in other phases of the life cycle.

Evolution of the isoprene biosynthetic pathway in kudzu.

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

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.

An Arabidopsis histidine kinase is essential for megagametogenesis.

Cytokinin-Independent 1 (CKI1) belongs to a group of putative plant histidine kinases whose members do not appear to act as ethylene receptors. The deduced protein structure, combined with the observation that Arabidopsis callus cultures overexpressing CKI1 exhibit a "cytokinin-independent" cell division and greening phenotype, led to the hypothesis that CKI1 is involved in cytokinin signaling, perhaps acting as a cytokinin receptor. To test the function of CKI1, we used a reverse-genetic approach to identify plants carrying T-DNA insertions in CKI1. Two independent alleles were identified, which produce the same developmental phenotype. Analyses of populations segregating for the cki1-5 or cki1-6 T-DNA insertion alleles failed to reveal any homozygous cki1 plants, indicating that the homozygous mutant condition was lethal. Based on segregation distortion, transmission studies, a microscopy-based examination of developing female gametophytes, and mRNA expression data, we suggest that CKI1 function is required for megagametophyte development. Our work with CKI1 mutants indicates that signal transduction by means of a HisAsp phosphorelay system may play an important and previously unsuspected role in female gametophyte development in Arabidopsis.

Effect of regulated overexpression of the MADS domain factor AGL15 on flower senescence and fruit maturation.

We have examined the effect of regulated overexpression of AGL15, a member of the MADS domain family of regulatory factors, on reproductive tissues. Using molecular and physiological markers, we show that constitutive overexpression of AGL15 in Arabidopsis leads to delay and down-regulation of senescence programs in perianth organs and developing fruits and alters the process of seed desiccation. Through genetic crosses, we show that the rate of water loss in the maturing seeds is dictated by the genetic composition and physiological state of the maternal tissue, rather than the embryo. To define the developmental time and/or place when senescence programs are most affected by elevated AGL15 levels, we expressed AGL15 under the control of various promoters. Expression during senescence or in abscission zone cells did not produce delays in floral organ senescence or abscission. Using a glucocorticoid-inducible expression system, we show that an increase in AGL15 levels around the time of flower opening is necessary to delay senescence and increase floral organ longevity.