Putative inorganic carbon transport and accumulation systems associated with the CO2 concentrating mechanism in Chlamydomonas reinhardtii

The CO 2 -concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon transporters, while specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii . Here, we have investigated whether the limiting-CO2 -inducible, putative ABC-type transporter, HLA3 might function as a HCO3 - transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity and [14C]-Ci uptake in very low CO2 conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. The data provide compelling evidence that HLA3 is directly or indirectly involved in HCO3 - transport and provide additional evidence supporting a role for LCIA in chloroplast envelope HCO3 - transport and for LCIB in chloroplast Ci accumulation. To further elucidate the function of LCIB, we identified two ad1 suppressors that can grow in low- CO2 but die in very low- CO2. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of low- CO2 acclimated suppressors under acclimation CO2 concentrations were more than two fold higher than ad1, apparently resulting from a more than 20 fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. We conclude that LCIB functions downstream of CAH3 in the CCM and probably plays a role in trapping CO2 released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO2 to support growth in low- CO2 acclimated cells, but not in very low- CO2 acclimated cells, even in the absence of LCIB

Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia

A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence

17 Pags.- 7 Figs. © 2023 The AuthorsLegumes establish symbioses with rhizobia by forming nitrogen-fixing nodules. Nitrate is amajor environmental factor that affects symbiotic functioning. However, the molecularmechanism of nitrate-induced nodule senescence is poorly understood. Comparative transcriptomic analysis reveals an NAC-type transcription factor inLotus japo-nicus, LjNAC094, that acts as a positive regulator in nitrate-induced nodule senescence.Stable overexpression and mutant lines ofNAC094were constructed and used for phenotypiccharacterization. DNA-affinity purification sequencing was performed to identify NAC094targeting genes and results were confirmed by electrophoretic mobility shift and transactiva-tion assays. Overexpression ofNAC094induces premature nodule senescence. Knocking outNAC094partially relieves nitrate-induced degradation of leghemoglobins and abolishes nodule expres-sion of senescence-associated genes (SAGs) that contain a conserved binding motif forNAC094. Nitrate-triggered metabolic changes in wild-type nodules are largely affected innac094mutant nodules. Induction ofNAC094and its targetingSAGswas almost blocked inthe nitrate-insensitivenlp1,nlp4, andnlp1 nlp4mutants. We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate-induced expression ofSAGs. Our study fills in a key gap between nitrate and the execution ofnodule senescence, and provides a potential strategy to improve nitrogen fixation and stresstolerance of legumes.This work was supported by the National Natural Science Foundation of China (32000192, 31870220), the Foundation of Hubei Hongshan Laboratory (2022hszd014), Fundamental Research Funds for the Central Universities (2662020SKPY007), and MCIN/AEI/10.13039/501100011033 (grant PID2020-113985GB-I00). We also thank the BaiChuan fellowship of College of Life Science and Technology, Huazhong Agricultural University, for funding support.Peer reviewe

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules

18 Pags.- 8 Figs. © 2023 The Authors. New Phytologist.Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.This work was supported by the Ministry of Science and Technology of the People’s Republic of China (2021YFA0910800), National Natural Science Foundation of China (31870220), the Foundation of Hubei Hongshan Laboratory (2022hszd014), HZAU-AGIS Cooperation Fund (SZYJY2022005), and MCIN/AEI/10.13039/ 501100011033 of Spain (grant PID2020-113985GB-I00).Peer reviewe

Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

2 Pags.- 1 Fig. © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.Excessive application of nitrogen fertilizers has inevitably resultedin environmental problems. The symbiotic nitrogen fixation (SNF) that occurs in the root nodules of leguminous plants provides asustainable source of reduced nitrogen in agricultural ecosystems. More than 200 genes have been reported to regulate SNF, including rhizobial infection, nodule organogenesis and senescence (Royet al., 2020). Mature nodules consist mainly of twocell types: infected cells (IC) that contain nitrogen-fixing bac-teroids and uninfected cells (UC) that mediate active metabolismand nutrient transport. Although it is well known that SNFrequires functional specialization, the specific genes responsiblefor transcriptional regulation and carbon/nitrogen metabolismand transport in IC and UC remain largely unexplored.Single-cell transcriptomics has emerged as a powerful tech-nique for investigating spatiotemporal patterns of gene expression.This work was supported by the National Natural Science Foundation of China (31870220, 32000192), the China Post-doctoral Science Foundation (2020M680103), Fundamental Research Funds for the Central Universities 2662020SKPY007 and MCIN/AEI/10.13039/501100011033 (grant PID2020-113985GB-I00)Peer reviewe

Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1

Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin

Putative inorganic carbon transport and accumulation systems associated with the CO2 concentrating mechanism in Chlamydomonas reinhardtii

The CO 2 -concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon transporters, while specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii . Here, we have investigated whether the limiting-CO2 -inducible, putative ABC-type transporter, HLA3 might function as a HCO3 - transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity and [14C]-Ci uptake in very low CO2 conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. The data provide compelling evidence that HLA3 is directly or indirectly involved in HCO3 - transport and provide additional evidence supporting a role for LCIA in chloroplast envelope HCO3 - transport and for LCIB in chloroplast Ci accumulation. To further elucidate the function of LCIB, we identified two ad1 suppressors that can grow in low- CO2 but die in very low- CO2. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of low- CO2 acclimated suppressors under acclimation CO2 concentrations were more than two fold higher than ad1, apparently resulting from a more than 20 fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. We conclude that LCIB functions downstream of CAH3 in the CCM and probably plays a role in trapping CO2 released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO2 to support growth in low- CO2 acclimated cells, but not in very low- CO2 acclimated cells, even in the absence of LCIB.</p

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii1[C][OA]

An active CO2-concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO2 (L-CO2; air level; approximately 0.04% CO2) or very low-CO2 (VL-CO2; approximately 0.01% CO2) conditions. A mutant, ad1, which is defective in the limiting-CO2-inducible, plastid-localized LCIB, can grow in high-CO2 or VL-CO2 conditions but dies in L-CO2, indicating a deficiency in a L-CO2-specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO2 but die in VL-CO2. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO2-acclimated suppressors under acclimation CO2 concentrations were more than 2-fold higher than ad1, apparently resulting from a more than 20-fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. However, photosynthetic rates of VL-CO2-acclimated cells under acclimation CO2 concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO2-concentrating mechanism and probably plays a role in trapping CO2 released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO2 to support growth in L-CO2-acclimated cells, but not in VL-CO2-acclimated cells, even in the absence of LCIB