The Stromal Processing Peptidase of Chloroplasts is Essential in Arabidopsis, with Knockout Mutations Causing Embryo Arrest after the 16-Cell Stage

Stromal processing peptidase (SPP) is a metalloendopeptidase located in the stroma of chloroplasts, and it is responsible for the cleavage of transit peptides from preproteins upon their import into the organelle. Two independent mutant Arabidopsis lines with T-DNA insertions in the SPP gene were analysed (spp-1 and spp-2). For both lines, no homozygous mutant plants could be detected, and the segregating progeny of spp heterozygotes contained heterozygous and wild-type plants in a ratio of 2∶1. The siliques of heterozygous spp-1 and spp-2 plants contained many aborted seeds, at a frequency of ∼25%, suggesting embryo lethality. By contrast, transmission of the spp mutations through the male and female gametes was found to be normal, and so gametophytic effects could be ruled out. To further elucidate the timing of the developmental arrest, mutant and wild-type seeds were cleared and analysed by Nomarski microscopy. A significant proportion (∼25%) of the seeds in mutant siliques exhibited delayed embryogenesis compared to those in wild type. Moreover, the mutant embryos never progressed normally beyond the 16-cell stage, with cell divisions not completing properly thereafter. Heterozygous spp mutant plants were phenotypically indistinguishable from the wild type, indicating that the spp knockout mutations are completely recessive and suggesting that one copy of the SPP gene is able to produce sufficient SPP protein for normal development under standard growth conditions

Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis

ALBINO3 (ALB3) is a well-known component of a thylakoid protein-targeting complex that interacts with the chloroplast signal recognition particle (cpSRP) and the cpSRP receptor, chloroplast filamentous temperature-sensitive Y (cpFtsY). Its protein-inserting function has been established mainly for light-harvesting complex proteins, which first interact with the unique chloroplast cpSRP43 component and then are delivered to the ALB3 integrase by a GTP-dependent cpSRP-cpFtsY interaction. In Arabidopsis (Arabidopsis thaliana), a subsequently discovered ALB3 homolog, ALB4, has been proposed to be involved not in light-harvesting complex protein targeting, but instead in the stabilization of the ATP synthase complex. Here, however, we show that ALB3 and ALB4 share significant functional overlap, and that both proteins are required for the efficient insertion of cytochrome f and potentially other subunits of pigment-bearing protein complexes. Genetic and physical interactions between ALB4 and ALB3, and physical interactions between ALB4 and cpSRP, suggest that the two ALB proteins may engage similar sets of interactors for their specific functions. We propose that ALB4 optimizes the insertion of thylakoid proteins by participating in the ALB3-cpSRP pathway for certain substrates (e.g. cytochrome f and the Rieske protein). Although ALB4 has clearly diverged from ALB3 in relation to the partner-recruiting C-terminal domain, our analysis suggests that one putative cpSRP-binding motif has not been entirely lost

Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis

ALBINO3 (ALB3) is a well-known component of a thylakoid protein-targeting complex that interacts with the chloroplast signal recognition particle (cpSRP) and the cpSRP receptor, chloroplast filamentous temperature-sensitive Y (cpFtsY). Its protein-inserting function has been established mainly for light-harvesting complex proteins, which first interact with the unique chloroplast cpSRP43 component and then are delivered to the ALB3 integrase by a GTP-dependent cpSRP-cpFtsY interaction. In Arabidopsis (Arabidopsis thaliana), a subsequently discovered ALB3 homolog, ALB4, has been proposed to be involved not in light-harvesting complex protein targeting, but instead in the stabilization of the ATP synthase complex. Here, however, we show that ALB3 and ALB4 share significant functional overlap, and that both proteins are required for the efficient insertion of cytochrome f and potentially other subunits of pigment-bearing protein complexes. Genetic and physical interactions between ALB4 and ALB3, and physical interactions between ALB4 and cpSRP, suggest that the two ALB proteins may engage similar sets of interactors for their specific functions. We propose that ALB4 optimizes the insertion of thylakoid proteins by participating in the ALB3-cpSRP pathway for certain substrates (e.g. cytochrome f and the Rieske protein). Although ALB4 has clearly diverged from ALB3 in relation to the partner-recruiting C-terminal domain, our analysis suggests that one putative cpSRP-binding motif has not been entirely lost

Control of PHERES1 Imprinting in Arabidopsis by Direct Tandem Repeats

ABSTRACT Genomic imprinting is an epigenetic phenomenon that causes monoallelic expression of specific genes dependent on the parent-of-origin. Imprinting of the Arabidopsis gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group complex as well as a distally located methylated region containing a tandem triple repeat sequence. In this study, we investigated the regulation of the close PHERES1 homolog PHERES2. We found that PHERES2 is also a direct target gene of the FIS Polycomb group complex, but, in contrast to PHERES1, PHERES2 is equally expressed from maternal and paternal alleles. Thus, PHERES2 is not regulated by genomic imprinting, correlating with the lack of tandem repeats at PHERES2. Eliminating tandem repeats from the PHERES1 locus abolishes PHERES1 imprinting, demonstrating that tandem repeats are essential for PHERES1 imprinting. Taking these results together, our study shows that the recently duplicated genes PHERES1 and PHERES2 are both target genes of the FIS Polycomb group complex but only PHERES1 is regulated by genomic imprinting, which is likely caused by the presence of repeat sequences in the proximity of the PHERES1 locus

Confined Water Dissociation in Microporous Defective Silicates: Mechanism, Dipole Distribution, and Impact on Substrate Properties

Interest in microporous materials has risen in recent years, as they offer a confined environment that is optimal to enhance chemical reactions. Calcium silicate hydrate (C-S-H) gel, the main component of cement, presents a layered structure with sub-nanometer-size disordered pores filled with water and cations. The size of the pores and the hydrophilicity of the environment make C-S-H gel an excellent system to study the possibility of confined water reactions. To investigate it, we have performed molecular dynamics simulations using the ReaxFF force field. The results show that water does dissociate to form hydroxyl groups. We have analyzed the water dissociation mechanism, as well as the changes in the structure and water affinity of the C-S-H matrix and water polarization, comparing the results with the behavior of water in a defective zeolite. Finally, we establish a relationship between water dissociation in C-S-H gel and the increase of hardness due to a transformation from a two- to a three-dimensional structure

New Suppressors of the Chloroplast Protein Import Mutant tic40 Reveal a Genetic Link between Protein Import and Thylakoid Biogenesis

To extend our understanding of chloroplast protein import and the role played by the import machinery component Tic40, a genetic screen for suppressors of chlorotic tic40 knockout mutant Arabidopsis plants was performed. As a result, two suppressor of tic40 loci, stic1 and stic2, were identified and characterized. The stic1 locus corresponds to the gene ALB4, which encodes a paralog of the well-known thylakoid protein targeting factor ALB3. The stic2 locus identified a previously unknown stromal protein that interacts physically with both ALB4 and ALB3. Genetic studies showed that ALB4 and STIC2 act together in a common pathway which also involves cpSRP54 and cpFtsY. Thus, we conclude that ALB4 and STIC2 both participate in thylakoid protein targeting, potentially for a specific subset of thylakoidal proteins, and that this targeting pathway becomes disadvantageous to the plant in the absence of Tic40. As the stic1 and stic2 mutants both suppressed tic40 specifically (other TIC-related mutants were not suppressed), we hypothesize that Tic40 is a multifunctional protein that, in addition to its originally-described role in protein import, is able to influence downstream processes leading to thylakoid biogenesis

New Suppressors of the Chloroplast Protein Import Mutant tic40 Reveal a Genetic Link between Protein Import and Thylakoid Biogenesis

To extend our understanding of chloroplast protein import and the role played by the import machinery component Tic40, a genetic screen for suppressors of chlorotic tic40 knockout mutant Arabidopsis plants was performed. As a result, two suppressor of tic40 loci, stic1 and stic2, were identified and characterized. The stic1 locus corresponds to the gene ALB4, which encodes a paralog of the well-known thylakoid protein targeting factor ALB3. The stic2 locus identified a previously unknown stromal protein that interacts physically with both ALB4 and ALB3. Genetic studies showed that ALB4 and STIC2 act together in a common pathway which also involves cpSRP54 and cpFtsY. Thus, we conclude that ALB4 and STIC2 both participate in thylakoid protein targeting, potentially for a specific subset of thylakoidal proteins, and that this targeting pathway becomes disadvantageous to the plant in the absence of Tic40. As the stic1 and stic2 mutants both suppressed tic40 specifically (other TIC-related mutants were not suppressed), we hypothesize that Tic40 is a multifunctional protein that, in addition to its originally-described role in protein import, is able to influence downstream processes leading to thylakoid biogenesis