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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]

Arbuscular mycorrhizal (AM) symbiosis is a widespread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3 , AMK8 , or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171 , the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation. [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]

Summary: The symbiotic interaction between arbuscular mycorrhizal fungi (AMF) and land plants is essential for efficient nutrient acquisition and utilisation. Our understanding of key processes controlling the AMF colonisation in rice is still limited.Dongxiang wild rice (DY) exhibited a stronger colonisation with Rhizophagus irregularis than the rice cultivar Zhongzao 35 (ZZ35). Chromosome segment substitution lines were constructed and the OsCERK1 gene from DY was mapped. Transgenic plants in the japonica rice Zhonghua 11 (ZZ11) were constructed to compare root colonisation by AMF.Chromosome single‐segment substitution lines containing OsCERK1DY showed higher phosphorus content and grain yield relative to ZZ35. Four amino acids substitutions were identified among the OsCERK1 haplotypes of DY, ZZ35 and ZH11 and two of these were in the second lysine‐motif domain, which is essential for the differences of AMF colonisation level among rice varieties. Heterologous expression of OsCERK1DY in ZH11 significantly enhanced AMF colonisation and increased resistance against the pathogenic fungi Magnaporthe oryzae. Notably, the OsCERK1DY haplotype was absent from 4660 cultivated rice varieties.We conclude that OsCERK1 is a key gene affecting the symbiotic interaction with AMF and OsCERK1DY has the biotechnological potential to increase rice phosphorus acquisition and utilisation efficiency for sustainable agriculture. See also the Commentary on this article by Lefebvre, 225: 1404–1406. [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]

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]