The effect of natural products used as pesticides on the soil microbiota: OECD 216 nitrogen transformation test fails to identify effects that were detected via q-PCR microbial abundance measurement.

BACKGROUND: Natural products present an environmentally attractive alternative to synthetic pesticides which have been implicated in the off-target effect. Currently, the assessment of pesticide toxicity on soil microorganisms relies on the OECD 216 N transformation assay (OECD stands for the Organisation Economic Co-operation and Development, which is a key international standard-setting organisation). We tested the hypotheses that (i) the OECD 216 assay fails to identify unacceptable effects of pesticides on soil microbiota compared to more advanced molecular and standardized tests, and (ii) the natural products tested (dihydrochalcone, isoflavone, aliphatic phenol, and spinosad) are less toxic to soil microbiota compared to a synthetic pesticide compound (3,5-dichloraniline). We determined the following in three different soils: (i) ammonium (NH4 +) and nitrate (NO3 -) soil concentrations, as dictated by the OECD 216 test, and (ii) the abundance of phylogenetically (bacteria and fungi) and functionally distinct microbial groups [ammonia-oxidizing archaea (AOA) and bacteria (AOB)] using quantitative polymerase chain reaction (q-PCR). RESULTS: All pesticides tested exhibited limited persistence, with spinosad demonstrating the highest persistence. None of the pesticides tested showed clear dose-dependent effects on NH4 + and NO3 - levels and the observed effects were <25% of the control, suggesting no unacceptable impacts on soil microorganisms. In contrast, q-PCR measurements revealed (i) distinct negative effects on the abundance of total bacteria and fungi, which were though limited to one of the studied soils, and (ii) a significant reduction in the abundance of both AOA and AOB across soils. This reduction was attributed to both natural products and 3,5-dichloraniline. CONCLUSION: Our findings strongly advocate for a revision of the current regulatory framework regarding the toxicity of pesticides to soil microbiota, which should integrate advanced and well-standardized tools. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco.

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co-factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF-deficient mutant plants, and we show that re-initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.

The plastid-encoded PsaI subunit stabilizes photosystem I during leaf senescence in tobacco.

PsaI is the only subunit of PSI whose precise physiological function has not yet been elucidated in higher plants. While PsaI is involved in PSI trimerization in cyanobacteria, trimerization was lost during the evolution of the eukaryotic PSI, and the entire PsaI side of PSI underwent major structural remodelling to allow for binding of light harvesting complex II antenna proteins during state transitions. Here, we have generated a tobacco (Nicotiana tabacum) knockout mutant of the plastid-encoded psaI gene. We show that PsaI is not required for the redox reactions of PSI. Neither plastocyanin oxidation nor the processes at the PSI acceptor side are impaired in the mutant, and both linear and cyclic electron flux rates are unaltered. The PSI antenna cross section is unaffected, state transitions function normally, and binding of other PSI subunits to the reaction centre is not compromised. Under a wide range of growth conditions, the mutants are phenotypically and physiologically indistinguishable from wild-type tobacco. However, in response to high-light and chilling stress, and especially during leaf senescence, PSI content is reduced in the mutants, indicating that the I-subunit plays a role in stabilizing PSI complexes.

Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light.

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild-type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non-photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild-type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light-regulated manner.

Accelerated electron transport from photosystem I to redox partners by covalently linked ferredoxin.

Photosystem I is a highly efficient and potent light-induced reductase that is considered to be an appealing target for integration into hybrid solar fuel production systems. However, rapid transport of multiple electrons from the reducing end of photosystem I to downstream processes in vivo is limited by the diffusion of its native redox partner ferredoxin that is a single electron carrier. Here, we describe the design and construction of a faster electron transfer interface based on anchoring ferredoxin to the reducing end of photosystem I thereby confining the diffusion space of ferredoxin to the near vicinity of its photosystem I binding and reduction site. This was achieved by fusing ferredoxin to the PsaE subunit of photosystem I by a flexible peptide linker and reconstituting PSI in vitro with the new fusion protein. A computational algorithm was developed in order to determine the optimal linker length that will confine ferredoxin to the vicinity of photosystem I's reducing end without restricting the formation of electron transfer complexes. According to the calculation, we reconstituted photosystem I with three fusion proteins comprising PsaE and ferredoxin separated by linkers of different lengths, namely 14, 19, and 25 amino acids, and tested their effect on electron transfer rates from photosystem I to downstream processes. Indeed, we found a significant enhancement of light dependent NADPH synthesis using photosystems containing the PsaE-ferredoxin fusion proteins, equivalent to a ten-fold increase in soluble ferredoxin concentration. We propose that such a system could be used for other ferredoxin dependent redox reactions, such as the enzymatic production of hydrogen, a promising alternative fuel. As the system is comprised entirely of natural amino acids and biological cofactors, it could be integrated into the energy conversion apparatus of photosynthetic organisms by genetic engineering.

A chloroplast light-regulated oxidative sensor for moderate light intensity in Arabidopsis.

The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.