Michael Hodges

Removal of bicarbonate from the non-heme iron at the acceptor side of photosystem II (PSII) has been shown to shift the midpoint potential of the primary quinone acceptor Q_A to a more positive value, thereby lowering the yield of singlet oxygen production and protecting PSII against photoinhibition. Previous studies have demonstrated that the properties of Q_A are altered in the Arabidopsis thaliana (Arabidopsis) photorespiratory mutant hrp1-1 that is deficient in peroxisomal hydroxypyruvate reductase 1 and accumulates glycolate in leaves (Messant et al. (2018) Plant Physiol. 177:1277–1285). We hypothesize that other photorespiratory mutants also show an alteration at the PSII acceptor side. To support this hypothesis, changes in PSII electron transfer were measured in three Arabidopsis photorespiratory cycle mutants: a knockout of 2-phosphoglycolate phosphatase 1 (pglp1-1), a knockout of the glycolate/glycerate transporter 1 (plgg1-1) and a knockdown of glycolate oxidase (amiRgox1gox2). Chlorophyll fluorescence decay kinetics were similar in all plant lines when grown in a high 10,000 ppm CO₂ condition that suppresses photorespiration. In contrast, when transferred from high to ambient CO₂, decay kinetics were slowed down in amiRgox1gox2, but not in pglp1-1 that accumulates 2-phosphoglycolate. On the other hand, plgg1-1 exhibited an alteration in decay kinetics only when ambient CO₂ was combined with high light exposure, a condition promoting photorespiration. Based on our present results we hypothesize that photorespiration can exert a regulatory effect on primary photosynthetic electron flow by replacing bicarbonate at the non-heme iron with glycolate.

Summary: Sustainable crop production demands solutions to reduce the overuse of synthetic nitrogen (N) fertilizers, and plant‐growth‐promoting bacteria offer a promising strategy by enhancing nutrient acquisition. This study investigated the ability of a nondiazotrophic bacterium, Enterobacter sp. SA187 (SA187), in enhancing Arabidopsis growth under low‐nitrate conditions and the underlying mechanisms. Arabidopsis seedlings were grown under different nitrate concentrations with or without SA187 inoculation. Growth traits were quantified alongside shoot and root nitrate and total N contents, and C : N ratios. Transcriptomic profiling (RNA‐seq) and qRT‐PCR were used to assess modified gene expression. Functional validation was conducted using ethylene‐insensitive ( ein2‐1 ) and high‐affinity nitrate transporter (HATS) mutants ( nrt2.5 , nrt2.6 ). SA187 significantly enhanced fresh weight, primary root length, and lateral root density under low nitrate, with benefits increasing as nitrate availability decreased. SA187 improved nitrate accumulation and shoot N allocation, reducing shoot C : N ratios. SA187 regulated expression of HATS and hormone‐responsive genes. The growth‐promoting effects were abolished in ein2‐1 , nrt2.5 , and nrt2.6 mutants, and SA187‐induced regulation of NRT2.5 occurred downstream of ethylene signaling, while NRT2.6 was partly ethylene independent. SA187 promotes growth under low nitrate possibly through ethylene‐mediated and HATS‐dependent reprogramming of nitrate accumulation and N allocation, supporting its use as a microbial solution for low‐input agriculture.

As atmospheric CO ₂ levels continue to increase, optimizing the CO ₂ fertilization effect which often falls short of its potential due to the physiological and metabolic limitations of plants becomes crucial. This study investigates the role of Enterobacter sp. SA187 (SA187), a plant growth-promoting bacterium, in enhancing growth and development of Arabidopsis thaliana under elevated atmospheric CO ₂ (eCO ₂ ) conditions. While SA187 inoculation did not have major effects under ambient CO ₂ , it was found to significantly enhance root and shoot biomass, and to increase N- and reduce C-contents under eCO ₂ . Moreover, transcriptomics and metabolomics suggested that SA187 modulated phytohormonal homeostasis, with activation of the salicylic acid, jasmonic acid and ethylene signalling pathways, and increased primary metabolism including the TCA cycle, N and carbohydrate metabolisms. Finally, the growth-promoting effects of SA187 were shown to be mediated through ethylene-dependent pathways, as evidenced with the ethylene-insensitive mutant ein2-1 which did not show similar benefits in plant fresh weight and altered gene expression. This beneficial plant-microbe interaction under eCO ₂ in a non-leguminous plant highlights a novel aspect of microbial influence on plant physiology in the context of climate change. These insights underscore the potential of utilizing SA187 to enhance plant performance and adaptability in future high CO ₂ environments, providing a sustainable approach to agricultural productivity as global CO ₂ levels increase.

Summary: Sustainable crop production demands solutions to reduce the overuse of synthetic nitrogen fertilizers, and plant-growth-promoting bacteria offer a promising strategy by enhancing nutrients acquisition. This study investigated ability of a non-diazotrophic bacterium, Enterobacter sp. SA187 (SA187), in enhancing Arabidopsis growth under low nitrate conditions and the underlying mechanisms. Arabidopsis seedlings were grown under different nitrate concentrations with or without SA187 inoculation. Growth traits were quantified alongside shoot and root nitrate and total nitrogen contents, and C:N ratios. Transcriptomic profiling (RNA-seq) and qRT-PCR were used to assess modified gene expression. Functional validation was conducted using ethylene-insensitive ( ein2-1 ) and high-affinity nitrate transporter (HATS) mutants ( nrt2.5, nrt2.6 ). SA187 significantly enhanced fresh weight, primary root length, and lateral root density under low nitrate, with benefits increasing as nitrate availability decreased. SA187 improved nitrate accumulation and shoot nitrogen allocation, reducing shoot C:N ratios. SA187 regulated expression of HATS and hormone-responsive genes. The growth-promoting effects were abolished in ein2-1, nrt2.5, and nrt2.6 mutants, and SA187-induced regulation of NRT2.5 occurred downstream of ethylene signaling, while NRT2.6 was partly ethylene-independent. SA187 promotes growth under low nitrate possibly through ethylene-mediated and HATS-dependent reprogramming of nitrate accumulation and nitrogen allocation, supporting its use as a microbial solution for low-input agriculture.

Photosynthesis—the conversion of energy from sunlight into chemical energy—is essential for life on Earth. Yet there is much we do not understand about photosynthetic energy conversion on a fundamental level: how it evolved and the extent of its diversity, its dynamics, and all the components and connections involved in its regulation. In this commentary, researchers working on fundamental aspects of photosynthesis including the light-dependent reactions, photorespiration, and C4 photosynthetic metabolism pose and discuss what they view as the most compelling open questions in their areas of research.

Besides the historical and traditional use of nuclear magnetic resonance (NMR) spectroscopy as a structure elucidation tool for proteins and metabolites, its quantification ability allows the determination of metabolite amounts and therefore enzymatic activity measurements. For this purpose, ¹H-NMR with adapted water pulse pre-saturation sequences and calibration curves with commercial standard solutions can be used to quantify the photorespiratory cycle intermediates, 2-phosphoglycolate and glycolate, associated with the phosphoglycolate phosphatase reaction. The intensity of the ¹H-NMR signal of glycolate produced by the activity of purified recombinant Arabidopsis thaliana PGLP1 can therefore be used to determine PGLP1 enzymatic activities and kinetic parameters.

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O₂ and releases H₂O₂. The oxidation of o-dianisidine by H₂O₂ catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni²⁺-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.