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Light is a critical environmental signal that is perceived by various photoreceptors and is of great significance in the photosynthetic growth of algae and plants. The phytochrome‐lacking model green alga Chlamydomonas reinhardtii possesses heme oxygenase (HMOX1) and phycocyanobilin ferredoxin oxidoreductase (PCYA1) to synthesize the linear tetrapyrrole bilin from heme in the chloroplast. The hmox1 mutant has photosynthetic growth deficiency and accumulation of photosystem I proteins such as LHCA1 is severely inhibited, and these defects could be rescued by exogenous bilin feeding in a blue light‐dependent manner. To investigate the contribution of the typical blue/ultraviolet light photoreceptors PHOT, aCRY, pCRY, and UVR8 in the process of bilin and blue light‐dependent recovery of LHCA1 protein in hmox1, we generated double mutants of these photoreceptors in hmox1, as well as a triple mutant of phot uvr8 hmox1, to analyze the LHCA1 protein abundance in these mutants. Results clearly showed that PHOT, CRYs, and UVR8 do not participate in this process. In addition, transcriptome profiling analysis of the hmox1 and its genetically complemented strain ho1C2 during dark‐to‐blue light transition revealed a total of 269 blue light‐responsive genes independent of bilin (|fold change| ≥ 2). RNA‐seq also identified a set of 249 differentially expressed genes that are dependent on both blue light and bilin. These findings provide valuable insights for elucidating the role of bilin in mediating blue light signaling pathways in Chlamydomonas. [ABSTRACT FROM AUTHOR]

Legumes establish symbiosis with rhizobia, forming nitrogen-fixing nodules. The central role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in nodule biology has been clearly established. Recently, hydrogen sulfide (H2S) and other reactive sulfur species (RSS) have emerged as novel signaling molecules in animals and plants. A major mechanism by which ROS, RNS, and RSS fulfil their signaling role is the post-translational modification of proteins. To identify possible functions of H2S in nodule development and senescence, we used the tag-switch method to quantify changes in the persulfidation profile of common bean (Phaseolus vulgaris) nodules at different developmental stages. Proteomic analyses indicate that persulfidation plays a regulatory role in plant and bacteroid metabolism and senescence. The effect of a H2S donor on nodule functioning and on several proteins involved in ROS and RNS homeostasis was also investigated. Our results using recombinant proteins and nodulated plants support a crosstalk among H2S, ROS, and RNS, a protective function of persulfidation on redox-sensitive enzymes, and a beneficial effect of H2S on symbiotic nitrogen fixation. We conclude that the general decrease of persulfidation levels observed in plant proteins of aging nodules is one of the mechanisms that disrupt redox homeostasis leading to senescence. [ABSTRACT FROM AUTHOR]

Meeting the ever-increasing food demands of a growing global population while ensuring resource and environmental sustainability presents significant challenges for agriculture worldwide. Arbuscular mycorrhizal symbiosis (AMS) has emerged as a potential solution by increasing the surface area of a plant's root system and enhancing the absorption of phosphorus, nitrogen nutrients, and water. Consequently, there is a longstanding hypothesis that rice varieties exhibiting more efficient AMS could yield higher outputs at reduced input costs, paving the way for the development of Green Super Rice (GSR). Our prior research study identified a variant, OsCERK1DY, derived from Dongxiang wild-type rice, which notably enhanced AMS efficiency in the rice cultivar "ZZ35." This variant represents a promising gene for enhancing yield and nutrient use efficiency in rice breeding. In this study, we conducted a comparative analysis of biomass, crop growth characteristics, yield attributes, and nutrient absorption at varying soil nitrogen levels in the rice cultivar "ZZ35" and its chromosome single-segment substitution line, "GJDN1." In the field, GJDN1 exhibited a higher AM colonization level in its roots compared with ZZ35. Notably, GJDN1 displayed significantly higher effective panicle numbers and seed-setting rates than ZZ35. Moreover, the yield of GJDN1 with 75% nitrogen was 14.27% greater than the maximum yield achieved using ZZ35. At equivalent nitrogen levels, GJDN1 consistently outperformed ZZ35 in chlorophyll (Chl) content, dry matter accumulation, major nutrient element accumulation, N agronomic efficiency (NAE), N recovery efficiency (NRE), and N partial factor productivity (NPFP). The performance of OsCERK1DY overexpression lines corroborated these findings. These results support a model wherein the heightened level of AMS mediated by OsCERK1DY contributes to increased nitrogen, phosphorus, and potassium accumulation. This enhancement in nutrient utilization promotes higher fertilizer efficiency, dry matter accumulation, and ultimately, rice yield. Consequently, the OsCERK1DY gene emerges as a robust candidate for improving yield, reducing fertilizer usage, and facilitating a transition towards greener, lower-carbon agriculture. [ABSTRACT FROM AUTHOR]

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: Legumes establish symbioses with rhizobia by forming nitrogen‐fixing nodules. Nitrate is a major environmental factor that affects symbiotic functioning. However, the molecular mechanism of nitrate‐induced nodule senescence is poorly understood.Comparative transcriptomic analysis reveals an NAC‐type transcription factor in Lotus japonicus, LjNAC094, that acts as a positive regulator in nitrate‐induced nodule senescence. Stable overexpression and mutant lines of NAC094 were constructed and used for phenotypic characterization. DNA‐affinity purification sequencing was performed to identify NAC094 targeting genes and results were confirmed by electrophoretic mobility shift and transactivation assays.Overexpression of NAC094 induces premature nodule senescence. Knocking out NAC094 partially relieves nitrate‐induced degradation of leghemoglobins and abolishes nodule expression of senescence‐associated genes (SAGs) that contain a conserved binding motif for NAC094. Nitrate‐triggered metabolic changes in wild‐type nodules are largely affected in nac094 mutant nodules. Induction of NAC094 and its targeting SAGs was almost blocked in the nitrate‐insensitive nlp1, nlp4, and nlp1 nlp4 mutants.We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate‐induced expression of SAGs. Our study fills in a key gap between nitrate and the execution of nodule senescence, and provides a potential strategy to improve nitrogen fixation and stress tolerance of legumes. [ABSTRACT FROM AUTHOR]

Summary: Legume nodules are symbiotic structures formed as a result of the interaction with rhizobia. Nodules fix atmospheric nitrogen into ammonia that is assimilated by the plant and this process requires strict metabolic regulation and signaling. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved as signal molecules at all stages of symbiosis, from rhizobial infection to nodule senescence. Also, reactive sulfur species (RSS) are emerging as important signals for an efficient symbiosis. Homeostasis of reactive molecules is mainly accomplished by antioxidant enzymes and metabolites and is essential to allow redox signaling while preventing oxidative damage. Here, we examine the metabolic pathways of reactive molecules and antioxidants with an emphasis on their functions in signaling and protection of symbiosis. In addition to providing an update of recent findings while paying tribute to original studies, we identify several key questions. These include the need of new methodologies to detect and quantify ROS, RNS, and RSS, avoiding potential artifacts due to their short lifetimes and tissue manipulation; the regulation of redox‐active proteins by post‐translational modification; the production and exchange of reactive molecules in plastids, peroxisomes, nuclei, and bacteroids; and the unknown but expected crosstalk between ROS, RNS, and RSS in nodules. [ABSTRACT FROM AUTHOR]

Rice domestication has dramatically improved its agronomic traits, albeit with unavoidable significantly reduced genetic diversity. Dongxiang common wild rice, the wild rice species distributed in northernmost China, exhibits excellent resistance against stress and diseases and provides a rich genetic resource for rice breeding. Most of the studies focus on the function of the plant genes, often disregarding the role of the root microbes associated with the plants. In this work, we isolated a Burkholderia strain from the root of Dongxiang wild rice, which we identified as Burkholderia cepacia BRDJ, based on a phylogenetic analysis. This strain promoted the rice growth under greenhouse conditions. The grain yield was higher in a rice line containing a small genomic fragment derived from the Dongxiang wild rice, compared to the indica rice cultivar Zhongzao 35. This new strain also increased the plant biomass under limiting nitrogen conditions. Interestingly, this strain had a differential effect on indica and japonica rice varieties under full nitrogen supply conditions. By genome sequencing and comparison with another two B. cepacia strains, we observed enriched genes related with nitrogen fixation and phytohormone and volatiles biosynthesis that may account for the growth-promoting effects of the BRDJ. BRDJ has the potential to be used as a biofertilizer in promoting nitrogen use efficiency and overall growth in rice. [ABSTRACT FROM AUTHOR]

Bilins are open-chain tetrapyrroles synthesized in phototrophs by successive enzymic reactions catalyzed by heme oxygenases (HMOXs/HOs) and ferredoxin-dependent biliverdin reductases (FDBRs) that typically serve as chromophore cofactors for phytochromes and phycobiliproteins. Chlamydomonas reinhardtii lacks both phycobiliproteins and phytochromes. Nonetheless, the activity and stability of photosystem I (PSI) and the catalytic subunit of magnesium chelatase (MgCh) named CHLH1 are significantly reduced and phototropic growth is significantly attenuated in a hmox1 mutant that is deficient in bilin biosynthesis. Consistent with these findings, previous studies on hmox1 uncovered an essential role for bilins in chloroplast retrograde signaling, maintenance of a functional photosynthetic apparatus, and the direct regulation of chlorophyll biosynthesis. In this study, we generated and screened a collection of insertional mutants in a hmox1 genetic background for suppressor mutants with phototropic growth restored to rates observed in wild-type 4A+ C. reinhardtii cells. Here, we characterized a suppressor of hmox1 named ho1su1 with phototrophic growth rates and levels of CHLH1 and PSI proteins similar to 4A+. Tetrad analysis indicated that a plasmid insertion co-segregated with the suppressor phenotype of ho1su1. Results from TAIL-PCR and plasmid rescue experiments demonstrated that the plasmid insertion was located in exon 1 of the HMOX1 locus. Heterologous expression of the bilin-binding reporter Nostoc punctiforme NpF2164g5 in the chloroplast of ho1su1 indicated that bilin accumulated in the chloroplast of ho1su1 despite the absence of the HMOX1 protein. Collectively, our study reveals the presence of an alternative bilin biosynthetic pathway independent of HMOX1 in the chloroplasts of Chlamydomonas cells. [ABSTRACT FROM AUTHOR]

The C[O.sub.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 (Ci; C[O.sub.2] and HC[O.sup.-.sub.3]) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-C[O.sub.2]--inducible, putative ABC-type transporter HLA3 might function as a HC[O.sup.-.sub.3] transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [[sup.14]C]-Ci uptake in very low C[O.sub.2] conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH--dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci--inducible plastid-localized protein required for normal Ci uptake or accumulation in low-C[O.sub.2] conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci--inducible plastid envelope protein reported to transport HC[O.sup.-.sub.3]) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HC[O.sup.-.sub.3] is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HC[O.sup.-.sub.3] transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HC[O.sup.-.sub.3] transport and a role for LCIB in chloroplast Ci accumulation. ABC transporter | C[O.sub.2]-concentrating mechanism | microalgae | RNAi

Summary: Light is the crucial environmental signal for desiccation‐tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin‐binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF‐containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin‐producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near‐UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first‐ever reported GAF‐only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual‐cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The chlorophyll biosynthesis regulator GENOMES UNCOUPLED 4 (GUN4) is conserved in nearly all oxygenic photosynthetic organisms. Recently, GUN4 has been found to be able to bind the linear tetrapyrroles (bilins) and stimulate the magnesium chelatase activity in the unicellular green alga Chlamydomonas reinhardtii. Here, we characterize GUN4 proteins from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 for their ability to bind bilins, and present the crystal structures of Synechocystis GUN4 in biliverdin‐bound, phycocyanobilin‐bound, and phytochromobilin‐bound forms at the resolutions of 1.05, 1.10, and 1.70 Å, respectively. These linear molecules adopt a cyclic‐helical conformation, and bind more tightly than planar porphyrins to the tetrapyrrole‐binding pocket of GUN4. Based on structural comparison, we propose a working model of GUN4 in regulation of tetrapyrrole biosynthetic pathway, and address the role of the bilin‐bound GUN4 in retrograde signaling. PDB Code(s): 7E2R, 7E2S, 7E2T and 7E2U; [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXa reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4: bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment. [ABSTRACT FROM AUTHOR]

The I FLU i overexpressing plants exhibited lower shoot fresh weight, produced fewer red nodules and more white nodules, and had lower haem content in the nodules (Figure 1m-o). Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules Keywords: haem; leghaemoglobin; legume nodules; single-cell transcriptomics; symbiotic nitrogen fixation EN haem leghaemoglobin legume nodules single-cell transcriptomics symbiotic nitrogen fixation 616 618 3 04/11/22 20220401 NES 220401 Excessive application of nitrogen fertilizers has inevitably resulted in environmental problems. [Extracted from the article]

Chloroplasts convert solar energy into biologically useful forms of energy by performing photosynthesis. Although light and particular genes are known to promote chloroplast development, little is known about the mechanisms that regulate the tissue‐specificity and cell‐specificity of chloroplast biogenesis. Thus, the mechanisms that determine whether non‐photosynthetic plastids rather than chloroplasts develop in petals remain largely unexplored. Although heat stress is known to inhibit photosynthesis, we do not know whether heat stress affects chloroplast biogenesis. Here, we report that heat stress up‐regulates the expression of chlorophyll biosynthesis‐related genes and promotes chloroplasts biogenesis in petals overexpressing SOC1 (suppressor of overexpression of CO) and novel SOC1‐like genes. We also found that these specific MADS‐box transcription factors are present in most photosynthetic eukaryotes and that the expression of more than one homolog is observed in chloroplast‐containing tissues. These findings not only provide novel insights into the tissue specificity of chloroplast biogenesis and a method for producing green petals but also are consistent with heat stress influencing chloroplast biogenesis in higher plants. Mechanisms that control the tissue specificity of chloroplast biogenesis are poorly understood. We found that overexpressing genes encoding particular MADS‐box transcription factors up‐regulate the expression of chlorophyll biosynthesis‐related genes and promote chloroplasts biogenesis in the petals of heat‐stressed plants. These findings provide novel insight into the mechanisms that control the tissue specificity of chloroplast biogenesis and a method for producing green petals. [ABSTRACT FROM AUTHOR]

Summary: Suppression of innate immunity is essential for rhizobial infection and colonization in compatible interactions with leguminous plants. In Medicago nad1 mutant plants, innate immunity is excessively activated, resulting in necrotic cell death after rhizobia are released from infection threads into symbiotic cells, suggesting that innate immunity plays a critical role in regulating bacteroid persistence. In this study, we identified three respiratory burst oxidase homologs (Rboh) and one calcium‐dependent protein kinase (CDPK) as key factors for the activation of immunity in Medicago nodules using genetic and biochemical methods. Knock‐out of either MtRbohB or MtRbohD in nad1‐1 mutant plants produced effective nodules with intact symbiotic cells, while knock‐out of MtRbohC decreased brown pigment deposition, leading to less necrosis in nad1‐1 mutant nodules. MtCDPK5 directly phosphorylated MtRbohB, MtRbohC and MtRbohD, which triggered immune responses in plants. Knock‐out of MtCDPK5 in nad1‐1 mutant plants partially restored nitrogen‐fixing nodules. Overexpression of the constitutively activated variant MtCDPK5VK under the control of the NAD1 promoter elicited strong immune responses, resulting in ineffective nodules in wild‐type plants. Our data provide direct evidence that host plants utilize innate immunity to regulate rhizobial colonization in symbiotic cells in Medicago truncatula. [ABSTRACT FROM AUTHOR]

Abstract: During the establishment of rhizobia‐legume symbiosis, the cytokinin receptor LHK1 (Lotus Histidine Kinase 1) is essential for nodule formation. However, the mechanism by which cytokinin signaling regulates symbiosis remains largely unknown. In this study, an LHK1‐interacting protein, LjCZF1, was identified and further characterized. LjCZF1 is a C3HC4‐type RING finger protein that is highly conserved in plants. LjCZF1 specifically interacted with LHK1 in yeast two‐hybrid, in vitro pull‐down and co‐immunoprecipitation assays conducted in tobacco. Phosphomimetic mutation of the potential threonine (T167D) phosphorylation site enhanced the interaction between LjCZF1 and LHK1, whereas phosphorylation mutation (T167A) eliminated this interaction. Transcript abundance of LjCZF1 was up‐regulated significantly after inoculation with rhizobia. The LORE1 insertion mutant and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐mediated knockout mutant Lotus japonicus plants demonstrated significantly reduced number of infection threads and nodules. In contrast, plants over‐expressing LjCZF1 exhibited increased numbers of infection threads and nodules. Collectively, these data support the notion that LjCZF1 is a positive regulator of symbiotic nodulation, possibly through interaction with LHK1. [ABSTRACT FROM AUTHOR]

Anoxygenic photosynthetic prokaryotes arose in ancient oceans ~3.5 billion years ago. The evolution of oxygenic photosynthesis by cyanobacteria followed soon after, enabling eukaryogenesis and the evolution of complex life. The Archaeplastida lineage dates back ~1.5 billion years to the domestication of a cyanobacterium. Eukaryotic algae have subsequently radiated throughout oceanic/freshwater/terrestrial environments, adopting distinctive morphological and developmental strategies for adaptation to diverse light environments. Descendants of the ancestral photosynthetic alga remain challenged by a typical diurnally fluctuating light supply ranging from ~0 to ~2000 μE m−2 s−1. Such extreme changes in light intensity and variations in light quality have driven the evolution of novel photoreceptors, light-harvesting complexes and photoprotective mechanisms in photosynthetic eukaryotes. This minireview focuses on algal light sensors, highlighting the unexpected roles for linear tetrapyrroles (bilins) in the maintenance of functional chloroplasts in chlorophytes, sister species to streptophyte algae and land plants. [ABSTRACT FROM AUTHOR]

The inducible CO 2 concentrating mechanism (CCM) of microalgae is essential for their acclimation to highly variable aquatic inorganic carbon levels. The key transcriptional regulator, CIA5, affects expression of thousands of genes in Chlamydomonas reinhardtii , but the molecular characteristics of this important protein are poorly understood. This study identifies a functional activation domain in CIA5 and demonstrates the functionality of a mini-CIA5 including only the zinc-binding domain and the identified activation domain. A highly conserved 130aa region from CIA5 exhibits auto-activation in yeast and appears responsible for the markedly slow migration of CIA5 when analyzed by SDS-PAGE. This 130aa region or either half of this region also effectively replaced the activation domain of a modified designer Transcription Activator-like Element (dTALE) in targeted activation of an endogenous Chlamydomonas gene. Additionally, a mini-CIA5 combining the conserved zinc-binding domain with the 130aa putative activation domain complemented the growth phenotype of the cia5 mutant and triggered CO 2 -regulated gene expression patterns similar to wild-type cells or cia5 complemented with the full-length CIA5 . Although the mini-CIA5 complementation did not fully restore wild-type growth rates or full gene induction/repression amplitudes, especially in very low CO 2 , this newly identified activation domain combined with the previously described zinc-binding domain are demonstrated to be the key essential components of CIA5 that permit rapid CIA5-mediated responses to changes in CO 2 concentrations. [ABSTRACT FROM AUTHOR]

The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia., Here, a Medicago truncatula nodules with activated defense 1 ( nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules., In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum., Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjβCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus. However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus. [ABSTRACT FROM AUTHOR]

Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume. [ABSTRACT FROM AUTHOR]

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1 -transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Bilins are open-chain tetrapyrroles synthesized in phototrophs by successive enzymic reactions catalyzed by heme oxygenases (HMOXs/HOs) and ferredoxin-dependent biliverdin reductases (FDBRs) that typically serve as chromophore cofactors for phytochromes and phycobiliproteins. Chlamydomonas reinhardtii lacks both phycobiliproteins and phytochromes. Nonetheless, the activity and stability of photosystem I (PSI) and the catalytic subunit of magnesium chelatase (MgCh) named CHLH1 are significantly reduced and phototropic growth is significantly attenuated in a hmox1 mutant that is deficient in bilin biosynthesis. Consistent with these findings, previous studies on hmox1 uncovered an essential role for bilins in chloroplast retrograde signaling, maintenance of a functional photosynthetic apparatus, and the direct regulation of chlorophyll biosynthesis. In this study, we generated and screened a collection of insertional mutants in a hmox1 genetic background for suppressor mutants with phototropic growth restored to rates observed in wild-type 4A+ C. reinhardtii cells. Here, we characterized a suppressor of hmox1 named ho1su1 with phototrophic growth rates and levels of CHLH1 and PSI proteins similar to 4A+. Tetrad analysis indicated that a plasmid insertion co-segregated with the suppressor phenotype of ho1su1 . Results from TAIL-PCR and plasmid rescue experiments demonstrated that the plasmid insertion was located in exon 1 of the HMOX1 locus. Heterologous expression of the bilin-binding reporter Nostoc punctiforme NpF2164g5 in the chloroplast of ho1su1 indicated that bilin accumulated in the chloroplast of ho1su1 despite the absence of the HMOX1 protein. Collectively, our study reveals the presence of an alternative bilin biosynthetic pathway independent of HMOX1 in the chloroplasts of Chlamydomonas cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Deng, Shi, Li, Larkin, Fan and Duanmu.)

The chlorophyll biosynthesis regulator GENOMES UNCOUPLED 4 (GUN4) is conserved in nearly all oxygenic photosynthetic organisms. Recently, GUN4 has been found to be able to bind the linear tetrapyrroles (bilins) and stimulate the magnesium chelatase activity in the unicellular green alga Chlamydomonas reinhardtii. Here, we characterize GUN4 proteins from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 for their ability to bind bilins, and present the crystal structures of Synechocystis GUN4 in biliverdin-bound, phycocyanobilin-bound, and phytochromobilin-bound forms at the resolutions of 1.05, 1.10, and 1.70 Å, respectively. These linear molecules adopt a cyclic-helical conformation, and bind more tightly than planar porphyrins to the tetrapyrrole-binding pocket of GUN4. Based on structural comparison, we propose a working model of GUN4 in regulation of tetrapyrrole biosynthetic pathway, and address the role of the bilin-bound GUN4 in retrograde signaling., (© 2021 The Protein Society.)

In almost all symbiotic interactions between rhizobia and leguminous plants, host flavonoid-induced synthesis of Nod factors in rhizobia is required to initiate symbiotic response in plants. In this study, we found that Lotus japonicus Nod factor receptor 5 (LjNFR5) might directly regulate flavonoid biosynthesis during symbiotic interaction with rhizobia. A yeast two-hybrid analysis revealed that a dihydroflavonol-4-reductase-like protein (LjDFL1) interacts with LjNFR5. The interaction between MtDFL1 and MtNFP, two Medicago truncatula proteins with homology to LjDFL1 and LjNFR5, respectively, was also shown, suggesting that interaction between these two proteins might be conserved in different legumes. LjDFL1 was highly expressed in root hairs and epidermal cells of root tips. Lotus ljdfl1 mutants and Medicago mtdfl1 mutants produced significantly fewer infection threads (ITs) than the wild-type control plants following rhizobial treatment. Furthermore, the roots of stable transgenic L. japonicus plants overexpressing LjDFL1 formed more ITs than control roots after exposure to rhizobia. These data indicated that LjDFL1 is a positive regulator of symbiotic signaling. However, the expression of LjDFL1 was suppressed by rhizobial treatment, suggesting that a negative feedback loop might be involved in regulation of the symbiotic response in L. japonicus.

Bilins are linear tetrapyrroles commonly used as chromophores of phycobiliproteins and phytochromes for light-harvesting or light-sensing in photosynthetic organisms. Many eukaryotic algae lack both phycobiliproteins and phytochromes, but retain the bilin biosynthetic enzymes including heme oxygenase (HO/HMOX) and ferredoxin-dependent biliverdin reductase (FDBR). Previous studies on Chlamydomonas reinhardtii heme oxygenase mutant ( hmox1 ) have shown that bilins are not only essential retrograde signals to mitigate oxidative stress during diurnal dark-to-light transitions, they are also required for chlorophyll accumulation and maintenance of a functional photosynthetic apparatus in the light. However, the underlying mechanism of bilin-mediated regulation of chlorophyll biosynthesis is unclear. In this study, Chlamydomonas phycocyanobilin:ferredoxin oxidoreductase PCYA1 FDBR domain was found to specifically interact with the rate-limiting chlorophyll biosynthetic enzyme LPOR (light-dependent protochlorophyllide oxidoreductase). PCYA1 is partially associated with chloroplast envelope membrane, consistent with the observed export of bilin from chloroplast to cytosol by cytosolic expression of a bilin-binding reporter protein in Chlamydomonas. Both the pcya1-1 mutant with the carboxyl-terminal extension of PCYA1 eliminated and efficient knockdown of PCYA1 expression by artificial microRNA exhibited no significant impact on algal phototrophic growth and photosynthetic proteins accumulation, indicating that the conserved FDBR domain is sufficient and minimally required for bilin biosynthesis and functioning. Taken together, these studies provide novel insights into the regulatory role of PCYA1 in chlorophyll biosynthesis via interaction with key Chl biosynthetic enzyme.

Nitrogen-fixing rhizobia have established a symbiotic relationship with the legume family through more than 60 million years of evolution. Hundreds of legume host genes are involved in the SNF (symbiotic nitrogen fixation) process, such as recognition of the bacterial partners, nodulation signaling and nodule development, maintenance of highly efficient nitrogen fixation within nodules, regulation of nodule numbers, and nodule senescence. However, investigations of SNF-related gene functions and dissecting molecular mechanisms of the complicated signaling crosstalk on a genomic scale were significantly restricted by insufficient mutant resources of several representative model legumes. Targeted genome-editing technologies, including ZFNs, TALENs, and CRISPR-Cas systems, have been developed in recent years and rapidly revolutionized biological research in many fields. These technologies were also applied to legume plants, and significant progress has been made in the last several years. Here, we summarize the applications of these genome-editing technologies, especially CRISPR-Cas9, toward the study of SNF in legumes, which should greatly advance our understanding of the basic mechanisms underpinning the legume-rhizobia interactions and guide the engineering of the SNF pathway into nonlegume crops to reduce the dependence on the use of nitrogen fertilizers for sustainable development of modern agriculture., (© 2017 Elsevier Inc. All rights reserved.)

The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.