Abstract Arabidopsis thaliana ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) interacts with peroxisomal membrane-bound ASCORBATE PEROXIDASE3 (APX3). This interaction involves the C-terminal sequence of APX3 (i.e., a transmembrane domain plus a few basic amino acid residues). The specificity of the AKR2A–APX3 interaction suggests that AKR2A may function as a molecular chaperone for APX3 because binding of AKR2A to the transmembrane domain can prevent APX3 from forming aggregates after translation. Analysis of three akr2a mutants indicates that these mutant plants have reduced steady state levels of APX3. Reduced expression of AKR2A using RNA interference also leads to reduced steady state levels of APX3 and reduced targeting of APX3 to peroxisomes in plant cells. Since AKR2A also binds specifically to the chloroplast OUTER ENVELOPE PROTEIN7 (OEP7) and is required for the biogenesis of OEP7, AKR2A may serve as a molecular chaperone for OEP7 as well. The pleiotropic phenotype of akr2a mutants indicates that AKR2A plays many important roles in plant cellular metabolism and is essential for plant growth and development.
Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.
An underlying mechanism for reductions in crop yield under stress conditions is excessive production of reactive oxygen species (ROS) that can damage lipids, nucleic acids, and proteins, leading to disruption of physiological processes. The aim of this study was to determine whether overexpression of the gene for a peroxisomal antioxidant enzyme, ascorbate peroxidase 3 (APX3), could provide protection of photosynthesis during drought when the potential rises for excessive photorespiratory H 2 O 2 production. Tobacco ( Nicotiana tabacum L.) plants were transformed to constitutively overexpress the Arabidopsis thaliana gene for APX3. Following repeated water‐deficit cycles, fruit number and seed mass of transgenic plants were significantly higher than those of control plants. In another experiment, water deficit was developed gradually by reducing, in stages, the extent to which water lost was replenished. Genotypic differences in gas‐exchange parameters were observed at the 25% replenishment stage and at 5 h after severely stressed plants were rewatered. At these times, transgenic plants exhibited greater rates of CO 2 assimilation ( A ), stomatal conductance ( g s ), and internal CO 2 (C i ) to atmospheric CO 2 (C a ) concentration than control plants, suggesting that differences in A were controlled by differences in g s Although these data did not support the idea that overexpression of the gene for APX3 enhances protection of the photosynthetic apparatus during water deficit, overexpression of APX3 may affect other cellular metabolisms that result in higher A under moderate water‐deficit conditions and therefore higher seed mass after repeated water‐deficit treatments.
Summary The Arabidopsis ankyrin repeat‐containing protein AKR2 was identified as a GF14λ‐interacting protein in a yeast two‐hybrid screening (GF14λ is a 14‐3‐3 protein). Reduced expression of AKR2 by using the antisense technique results in small necrotic areas in leaves accompanied by higher production of H 2 O 2 , similar to the hypersensitive response to pathogen infection in plant disease resistance. Transcripts of genes encoding pathogen‐induced protein PR‐1 (pathogen‐related protein 1) and stress‐responsive protein GST6 (glutathione S‐transferase 6) are increased in antisense plants. The resistance to a bacterial pathogen infection was also increased by at least 10‐fold in antisense plants. AKR2 also interacts with another GF14λ‐interacting protein, the ascorbate peroxidase 3 that scavenges H 2 O 2 in plant cells. These data suggest that AKR2 may be a negative regulator of PR‐1 expression, and is probably involved in the regulation of antioxidation metabolism that is shared by both disease resistance and stress responses.
The Arabidopsis gene GF14λ that encodes a 14-3-3 protein was introduced into cotton plants to explore the physiological roles that GF14λ might play in plants. The expression level of GF14λ under the control of the cauliflower mosaic virus 35S promoter varied in transgenic cotton plants, and lines that expressed GF14λ demonstrated a "stay-green" phenotype and improved water-stress tolerance. These lines wilted less and maintained higher photosynthesis than segregated non-transgenic control plants under water-deficit conditions. Stomatal conductance appears to be the major factor for the observed higher photosynthetic rates under water-deficit conditions. The stomatal aperture of transgenic plants might be regulated by GF14λ through some transporters such as H+-ATPase whose activities are controlled by their interaction with 14-3-3 proteins. However, since 14-3-3 proteins interact with numerous proteins in plant cells, many metabolic processes could be affected by the GF14λ overexpression. Whatever the mechanisms, the traits observed in the GF14λ-expressing cotton plants are beneficial to crops under certain water-deficit conditions.
Summary Animal CHIP proteins are chaperone‐dependent E3 ubiquitin ligases that physically interact with Hsp70, Hsp90 and proteasome, promoting degradation of a selective group of non‐native or damaged proteins in animal cells. The plant CHIP‐like protein, AtCHIP, also plays important roles in protein turnover metabolism. AtCHIP interacts with a proteolytic subunit, ClpP4, of the chloroplast Clp protease in vivo , and ubiquitylates ClpP4 in vitro . The steady‐state level of ClpP4 is reduced in AtCHIP ‐overexpressing plants under high‐intensity light conditions, suggesting that AtCHIP targets ClpP4 for degradation and thereby regulates the Clp proteolytic activity in chloroplasts under certain stress conditions. Overexpression of ClpP4 in Arabidopsis leads to chlorotic phenotypes in transgenic plants, and chloroplast structures in the chlorotic tissues of ClpP4 ‐overexpressing plants are abnormal and largely devoid of thylakoid membranes, suggesting that ClpP4 plays a critical role in chloroplast structure and function. As AtCHIP is a cytosolic protein that has been shown to play an important role in regulating an essential chloroplast protease, this research provides new insights into the regulatory networks controlling protein turnover catabolism in chloroplasts.
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