BackgroundSalinity stress is a major global challenge that impairs plant growth by disrupting numerous physiological and biochemical processes. Nano-fertilizers, particularly green synthesized nanoparticles, offer a promising and eco-friendly strategy to enhance plant resilience and productivity under such stress. Accordingly, this study addresses a significant research gap by exploring the potential role of Molybdenum trioxide nanoparticles (MoO₃NPs) in improving salinity tolerance in molokhia, a topic that has received limited attention in previous studies.ResultsMolybdenum trioxide nanoparticles (MoO₃NPs) were fabricated using Medicago polymorpha fruit extract and characterized as spherical, orthorhombic α-MoO₃ with an average size of 9.2 ± 2.7 nm. Molokhia (Corchorus olitorius L.) was foliar-sprayed with MoO₃NPs at various dosages (0, 25, 50, and 100 mg/L) to mitigate the harmful effects of salinity stress (250 mM NaCl) on 30-day-old Molokhia plants. Application of 250 mM NaCl significantly inhibited growth, photosynthetic pigments, antioxidant activity, and osmolyte accumulation, alongside increased oxidative stress markers (MDA and H2O2). Foliar spraying with biogenic MoO3NPs under saline conditions, especially at 50 mg/L, effectively mitigated salt-induced damage by enhancing growth parameters, restoring photosynthetic pigment levels, and improving osmotic adjustment by boosting proline and soluble protein levels compared with control. Moreover, MoO3NPs treatment elevated enzymatic and non-enzymatic antioxidants (phenolics, flavonoids, glutathione, and ascorbic acid) and upregulated key stress-responsive genes (phenylalanine (PAL), magnesium chelatase (CHLH), superoxide dismutase1 (SOD1), catalase2 (CAT2), and flavonol synthase (FLS)), thereby reducing oxidative stress markers. These effects mitigated ROS accumulation, improved cellular stability, and enhanced overall plant resilience.ConclusionThese findings highlight that green-synthesized MoO₃NPs act as efficient nanofertilizers that alleviate salinity stress by promoting growth, reinforcing antioxidant defenses, and modulating stress-related genes. This dual role not only enhances crop resilience but also enriches the medicinal and nutritional value of Molokhia.

CsMORF9.3 regulates chlorophyll biosynthesis, affecting the RNA editing efficiency of matK-445, rpoA-200, ndhD-674 and ndhD-1310, and it interacts with CsMORFs, CsPPRs and CsCHLD proteins. Leaf color is an important factor affecting tea quality as well as the growth and development. It has been reported that several genes and transcription factors participated in chlorophyll metabolism in tea plants (Camellia sinensis). However, the role of chloroplast RNA editing factors in chlorophyll biosynthesis in C. sinensis remains poorly understood. In this study, multiple dysregulated RNA editing sites in the chloroplast genome were identified from etiolation and albino tea cultivars. Multiple organellar RNA editing factor 9.3 (CsMORF9.3), a core RNA editing factor localized in the chloroplasts, was identified as a candidate regulator of leaf coloration. Antisense oligonucleotide (AsODN) and virus-induced gene silencing (VIGS) confirmed that suppressing CsMORF9.3 expression reduces chlorophyll content, downregulates genes involved in chlorophyll biosynthesis and chloroplast development, as well as disrupts chloroplast RNA editing. Protein-protein interaction assays confirmed that CsMORF9.3 could form both homodimers and heterodimers with itself or other MORF proteins through yeast two-hybrid (Y2H), luciferase complementation imaging (LCI) assays, and bimolecular fluorescence complementation (BiFC) assays. Moreover, CsMORF9.3 was found to interact with multiple PLS-type pentatricopeptide repeat (PPR) proteins, including CsCRR21, CsCRR28, CsOTP84, and CsLPA66, as well as with CsCHLD, a key subunit of magnesium chelatase in chlorophyll biosynthesis. Our study provides the protein interaction of CsMORF9.3, demonstrating its potential role in regulating RNA editing and chlorophyll biosynthesis in C. sinensis. These results broaden the understanding of the regulatory mechanisms of chlorophyll biosynthesis and provide new insights into the breeding of etiolation and albino tea plant germplasm.

Broccoli (Brassica oleracea var. italica) is highly nutritious, rich in vitamin C, glucosinolates, and minerals. However, its high postharvest respiratory rate leads to rapid quality deterioration, particularly chlorophyll degradation and yellowing under ambient conditions. In China, the lack of timely pre-cooling facilities exacerbates postharvest losses. Therefore, developing safe, effective and low-cost preservation methods for broccoli during transportation is of great practical importance. In this study, RNA sequencing was employed to analyze the effects of preharvest 1-methylcyclopropene (1-MCP) and postharvest pre-cooling combined with cold treatments on gene expression in broccoli. Transcriptome analysis revealed that both treatments significantly upregulated or maintained key genes involved in chlorophyll biosynthesis (e.g., Glutamyl-tRNA reductase (GluTR), porphobilinogen deaminase (PBGD), magnesium chelatase (MgCh)) and downregulated chlorophyll degradation-related genes (e.g., Chlorophyllase (CLH), pheophytinase (PPH), pheophorbide a oxygenase (PaO)), resulting in enhanced chlorophyll retention. Furthermore, chlorophyllide a oxygenase (CAO) was upregulated, while chlorophyll b reductase (CBR) was downregulated, suggesting modulation of the chlorophyll cycle. These findings elucidate the molecular mechanisms by which 1-MCP and pre-cooling combined with cold regulate chlorophyll metabolism, providing new insights into the gene regulatory network underlying the postharvest quality maintenance in broccoli.

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprise the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompasses most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (Potyvirus nicotianainsculpentis)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed us to obtain plants exhibiting a white phenotype with their four alleles edited through invitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings may be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

Fvchli deficiency impairs ABA-mediated stomatal closure and enhances susceptibility to Xanthomonas fragariae in strawberry.

Intercellular communication via plasmodesmata (PD) is essential for plant growth, development, and defense, yet its regulation remains poorly understood. We set out to identify the chloroplast retrograde signals that regulate intercellular trafficking via PD. Using a combination of Arabidopsis thaliana mutants and gene silencing in Nicotiana benthamiana, we found that the metabolites of the tetrapyrrole biosynthetic pathway, most likely heme, can act to modulate PD-mediated intercellular trafficking. Knockout or knockdown of GUN5, encoding the catalytic subunit of the magnesium chelatase, revealed a correlation between reduced levels of total heme and severely decreased intercellular trafficking. Further, plants with reduced expression of genes involved in chloroplast RNA processing were shown to have decreased total heme levels and decreased intercellular trafficking. Like with GUN5, reduced expression of FC2, but not FC1, led to reduced levels of total heme and drastically reduced intercellular trafficking. Thus, the results support a model where a specific pool of heme regulates plasmodesmata and intercellular trafficking. We also identified genes that are potentially regulated by the heme signal to modify plasmodesmal function. Together, these findings strengthen the link between chloroplasts and PD in coordinating intercellular communication for optimal plant development and resource allocation.

Mutation of a magnesium chelatase BrCHLD affects the chlorophyll content and magnesium chelatase activity in Chinese cabbage.

Grain weight is a major determinant of wheat (Triticum aestivum L.) yield and is controlled by quantitative trait loci (QTLs) derived from natural variations. Here, we report the cloning of a major yield QTL in bread wheat that functions in the chlorophyll biosynthesis pathway. We found that the natural variation of CHELATASE SUBUNIT I-A (CHLI-A), encoding a magnesium chelatase subunit, enhances chlorophyll content and photosynthesis rate, leading to increased leaf area and grain weight. Increasing CHLI-A expression increases grain weight, indicating that photosynthesis is a limiting factor of wheat yield potential. The CHLI-A gene is absent in a portion of elite varieties, suggesting the potential of using this gene to improve grain yield in wheat.

Agrobacterium-mediated transformation allowed the establishment of the VIGS system based on the TRV for specific marker genes, Cte-PDS and Cte-chlH, in C. tenuiflora, a hemiparasitic plant. Castilleja tenuiflora Benth. (Orobanchaceae) is a medicinal and hemiparasitic plant recognized for its specialized metabolism. Genetic transformation systems are valuable for gene function analysis and understanding the regulation of the biosynthetic pathways of bioactive molecules. Here, we present two efficient Agrobacterium-mediated transformation protocols, defined as "transformation by injection" (Ti-AI) and "transformation by cocultivation" (Tc-AII). For both methods, two strains of A. tumefaciens were used: PCH32 and C58C1. These strains harbored a binary expression vector containing β-glucuronidase (GUS) as a reporter gene. The putative transformants tested positive in the GUS histochemical staining assay, which was confirmed via PCR. Tc-AII showed a higher transformation efficiency (Ti-AI: 23 vs. Tc-AII: 65%) although Ti-AI generated the highest survival rate of the putative transformants (Ti-AI: 75 vs. Tc-AII: 37%). Tc-AII-C58C1 was used to evaluate a VIGS system based on pTRV. Vectors were constructed with phytoene desaturase (Cte-PDS) and protoporphyrin magnesium chelatase subunit H (Cte-chlH) genes. Successful VIGS was demonstrated by gene silencing observed across various growth stages of C. tenuiflora when pTRV2-CtePDS and pTRV2-CtechlH were used, indicating that systemic viral infection was achieved. The photobleaching phenotype was observed 32days after agro-infection and more effective with pTRV2-CtechlH (80% photobleaching) than with pTRV2-CtePDS (31% photobleaching). The phenotype of the silenced plants was significantly correlated with the downregulation of endogenous Cte-chlH and Cte-PDS (P ≤ 0.01). These results indicate that the use of Cc-AII-C58C1 and TRV-based VIGS with pTRV2-CtechlH provides a tool to facilitate research on the functions of genes of interest involved in biotechnological processes in C. tenuiflora, yielding more significant information about gene silencing and genetic transformation in a non-model hemiparasitic medicinal plant.

Leaf colour mutants are ideal germplasm resources for investigating the mechanisms of chlorophyll (Chl) synthesis, chloroplast development and photosynthesis. In this study, we obtained a yellow-leaf mutant, designated SN288-2. The variant presented a yellow-leaf phenotype and halted the development of chloroplasts at the seedling stage, with reduced accumulation of Chl. The yellow-leaf phenotype reverted to the normal phenotype in the wheat revival stage. In addition, the ratio of the crucial Chl precursors protoporphyrin IX (Proto IX) and Mg-protoporphyrin IX (Mg-Proto IX) was relatively high in yellow leaves. Bulked segregant analysis sequencing (BSA-Seq) revealed that the aberrant phenotype was controlled by two recessive genes located on chromosomes 7A and 7D, designated Y1-7A and Y2-7D, respectively. Subsequent research focused on Y1-7A. We identified TraesCS7A03G1163900 as a viable candidate for Y1-7A, encoding a major subunit of Mg-chelatase that is essential for Chl synthesis. Whole-genome resequencing and Sanger sequencing revealed a 5.3 kb deletion on the long arm of chromosome 7A in SN388-2 that encompasses the entire Y1-7A sequence. Quantitative real-time PCR (qRT-PCR) revealed that the Y1-7A gene was predominantly expressed in green tissues and that the encoded protein was localized within the chloroplast. Moreover, weighted gene coexpression network analysis (WGCNA) revealed a gene module associated with leaf development and Chl content restoration. Consequently, these results provide a new theory regarding the regulation of Chl synthesis and chloroplast development. Overall, the loss of Y1-7A impaired the function of Mg-chelatase and blocked the conversion of Proto IX to Mg-Proto IX.

Passion fruit (Passiflora edulis) is a perennial, woody, tropical vine. It produces edible round to oval fruit that is highly favored for its unique aroma and taste, as well as its richness in antioxidants, vitamins, and minerals. However, functional genomics studies of passion fruit are scarce, as simple and efficient genetic tools are lacking for this species. Here, we developed virus-mediated protein overexpression (VOX) and virus-induced gene silencing (VIGS) vectors based on the telosma mosaic virus (TelMV), an emerging potyvirus that infects passion fruit plants worldwide. This vector, designated pTelMV-GW, incorporates Gateway-compatible recombination sites for rapid gene cloning. Using this vector, we achieved systemic stable expression of 2 heterologous proteins in passion fruit: green fluorescent protein (GFP) and bacterial phytoene synthase (CrtB). Additionally, pTelMV-GW containing different GFP fragments also induced systemic gene silencing in GFP-transgenic Nicotiana benthamiana plants. Furthermore, we used this vector to trigger phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) silencing in passion fruit plants. The TelMV-based VIGS was enhanced using a mild TelMV strain encoding a mutated helper-component proteinase (HC-Pro) with impaired RNA silencing suppressor activity. This upgraded vector (pTelMV-R181K-GW), containing PDS or ChlI fragments, induced clear photobleaching or yellowing phenotypes in passion fruit plants. Overall, our work presents a set of VIGS and VOX vectors for use in passion fruit plants, a crucial step towards identifying horticulturally important genes for improving passion fruit production and quality.

Chrysanthemum (Chrysanthemum × morifolium Ramat.) is a widely cultivated ornamental species, among which green-flowered cultivar representing rarity and distinctiveness. However, the underlying mechanisms governing the formation and regulation of green color remain poorly understood. In this study, exposure to high-intensity light resulted in the loss of green coloration in green chrysanthemum, while low-intensity light preserved it during flower opening in chrysanthemum ‘Lv Dingdang’. A R1-type MYB transcription factor, CmREVEILLE2 (CmRVE2), was identified through transcriptomic analysis of flowers subjected to different light intensities (3,000 Lux for low light, LL; 6,000 Lux for moderate light, ML; and 12,000 Lux for high light, HL). CmRVE2 expression was significantly upregulated under HL conditions, while its expression was lower under LL compared to ML. Additionally, CmRVE2 was upregulated by abscisic acid (ABA) and downregulated by gibberellin (GA). Virus-induced gene silencing (VIGS) of CmRVE2 led to a marked increase in chlorophyll content and the upregulation of chlorophyll biosynthesis-related genes, while CmRVE2 overexpression resulted in the opposite trend. Yeast one-hybrid, electrophoretic mobility shift assays (EMSA), and transient activation assays demonstrated that CmRVE2 directly bound to the promoter of CHLI1, a gene encodes magnesium chelatase I subunit in chlorophyll biosynthesis. Furthermore, CmRVE2 repressed the expression of MAGNESIUM-PROTOPORPHYRIN IX MONOMETHYL ESTER OXIDATIVE CYCLASE1 (CRD1) and PROTOCHLOROPHYLLIDE OXIDOREDUCTASE1 (PORA1), thereby inhibiting chlorophyll synthesis. These findings provide new insights into how CmRVE2 mediates light signaling to negatively regulate chlorophyll biosynthesis in chrysanthemum flowers.

Filamentous temperature-sensitive H (FtsH) is a major protease for thylakoid protein quality control in photosynthetic organisms. Mutations of the AtFtsH2 subunit in Arabidopsis result in yellow variegated2 (var2) mutants. Genetic screening of ENHANCER OF VARIEGATION (EVR) loci provides new insights into the role of FtsH in chloroplast development beyond the degradation of photodamaged D1 protein. Here, using Arabidopsis, we uncover a novel function of EVR3, previously reported as ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN1 (EGY1), in regulating chlorophyll biosynthesis by association with the H subunit of magnesium chelatase (CHLH). Additionally, we identify a new var2 enhancer mutant, evr4-1, caused by a missense mutation in CHLH. The evr4-1 mutant shows a significant decline in accumulation of light-harvesting complexes rather than in photosystem II core proteins, while evr3 evr4 double mutants exhibit synthetic lethal phenotypes accompanied by a drastic reduction in the accumulation of chlorophyll and light-harvesting complexes. Furthermore, disruption of the thylakoid protein sorting pathway mediated by the chloroplast Signal Recognition Particle 54 kDa protein mitigates the chloroplast development defect in var2-4 evr4-1. Our findings underscore the critical role of thylakoid FtsH for thylakoid protein quality control when chlorophyll biosynthesis is disrupted.

Foxtail millet is a characteristic miscellaneous grain crop with many benefits in current agricultural production and is crucial in the adjustment of the planting structure and the sustainable development of dry farming. However, the harmful effects of weeds have become a critical challenge, restricting the modern production of foxtail millet. The effect of penoxsulam on the chlorophyll metabolism pathway of foxtail millet and its physiological mechanism was studied. Spraying penoxsulam on foxtail millet leaves significantly reduced the content of chlorophyll synthesis precursors (5-aminolevulinic acid (ALA), Porphobilinogen (PBG), Protoporphyrin IX (ProtoIX), Mg-protoporphyrin IX (Mg-ProtoIX), and Protochlorophyllide (Pchlide)). Moreover, the activities of key synthetic enzymes (magnesium chelatase (MgCh) decreased compared to control, while the activities of degrading enzymes (pheophorbide a oxygenase (PAO) and pheophytinase activities (PPH) increased significantly. The study revealed the mechanism of penoxsulam inducing crop phytotoxicity by interfering with the dynamic balance of chlorophyll metabolism, which provided a theoretical basis for the scientific application of herbicides and the study of foxtail millet drug resistance.

Shading alleviates chloroplastic photodamage of forcing cultivated tree peonies in spring management

Dichlormid protect wheat from fomesafen residual injury by increasing PPO expression and the photosynthesis characterize.

SummaryTomato fruit ripening is a complex developmental process that is important for fruit quality and shelf life. Many factors, including ethylene and several key transcription factors, have been shown to play important roles in the regulation of tomato fruit ripening. However, our understanding of the regulation of tomato fruit ripening is still limited. Here, we describe mut26, an EMS‐induced tomato (Solanum lycopersicum) mutant that exhibits chlorophyll‐deficient phenotypes in various organs, including fruits. Genetic mapping and functional analyses revealed that a single‐nucleotide substitution and a corresponding Pro398–>Ser mis‐sense mutation in SlChlH (GENOMES UNCOUPLED 5, GUN5), which encodes the H subunit of magnesium chelatase, are responsible for the defects in the mut26 strain. Transcript analyses towards the expression of many SlPhANGs revealed that mut26 is defective in plastid retrograde signalling during tomato fruit ripening initiation, namely the transition from mature green to breaker stage. mut26 exhibits delayed progression of fruit ripening characterized by reduced fruit ethylene emission, increased fruit firmness, reduced carotenoid content and delayed plastid conversion from chloroplast to chromoplast. Given that fruit ripening requires signalling from plastids to nucleus, these data support the hypothesis that GUN5‐mediated plastid retrograde signalling promotes tomato fruit ripening. We further showed that the delayed fruit ripening of mut26 is not likely caused by reduced chlorophyll content. Taken together, we identified a new function of SlChlH in the promotion of tomato fruit ripening and ethylene biosynthesis, suggesting that GUN5‐mediated plastid retrograde signalling plays a promotive role in tomato fruit ripening.

In the current study, the role of external applications of rutin (Rut) and silicon (Si) in stress tolerance was investigated. Although it is known that Si has a role in improving plant defense against a variety of stresses, the role of Rut application in stress response remains unclear. Therefore, the current study was designed to evaluate the function of the synergistic effect of combined Rut and Si applications on the photosynthetic capacity of maize seedlings under osmotic stress. Twenty-one-day-old seedlings were treated with Rut (60 mg L-1) and Si (1 mM), and exposed to osmotic stress (induced by 10% and 15% (w/v) polyethylene glycol) for 48 h. The individual application of Rut and Si and especially the simultaneous treatment of Rut+Si improved the gas exchange parameters, chlorophyll content, photosystem II (PSII) activity, Rubisco enzyme activity, and the expression levels of magnesium chelatase and Rubisco genes, but decreased the expression of chlorophyllase gene under osmotic stress in comparison to osmotic stress alone. These findings suggest that exogenous Rut and Si can improve photosynthetic capacity in maize seedlings exposed to osmotic stress by increasing PSII activity and the expression of genes involved in photosynthesis and chlorophyll metabolism, as well as reducing chlorophyll degradation. The simultaneous treatment of Rut+Si may be useful in developing osmotic stress tolerance of plants.

Chlorophylls and carotenoids are 2 pivotal photosynthetic pigments directly influencing the economic value of pepper (Capsicum annuum L.) fruits. However, the coordinated regulatory mechanisms governing the accumulation of both chlorophylls and carotenoids during pepper fruit development remain elusive. In this study, pepper B-box 10 (CaBBX10), a candidate hub transcription factor, was found to play dual roles in the early development of pepper fruit. CaBBX10 virus-induced gene silencing and overexpression experiments demonstrated that the encoded transcription factor promotes both chlorophyll and carotenoid accumulation in pepper fruit. Further comprehensive analyses showed that CaBBX10 directly binds to the promoter of magnesium chelatase subunit D subunit (CaCHLD) and phytoene synthase 1 (CaPSY1), thereby activating their expression in the chlorophyll and carotenoid biosynthesis pathways, respectively. Additionally, the photomorphogenic factor CaCOP1 was found to physically interact with CaBBX10 and lead to its degradation. Therefore, CaBBX10 may serve as a critical link connecting chlorophyll and carotenoid biosynthesis to light signaling. Altogether, our findings reveal a mechanism for the complex transcriptional regulation that simultaneously promotes chlorophyll and carotenoid accumulation in pepper fruit.

Mutations in TaCHLI impact chlorophyll levels and yield-related traits in wheat. Natural variations in TaCHLI-7A/B influence plant productivity, offering potential for molecular breeding. Chlorophyll is essential for plant growth and productivity. The CHLI subunit of the magnesium chelatase protein plays a key role inserting magnesium into protoporphyrin IX during chlorophyll biosynthesis. Here, we identify a novel wheat mutant chlorophyll (chl) that exhibits yellow-green leaves, reduced chlorophyll levels, and increased carotenoid content, leading to an overall decline in yield-related traits. Map-based cloning reveals that the chl phenotype is caused by a point mutation (Asp186Asn) in the TaCHLI-7D gene, which encodes subunit I of magnesium chelatase. Furthermore, the three TaCHLI mutants: chl-7b-1 (Pro82Ser), chl-7b-2 (Ala291Thr), and chl-7d-1 (Gly357Glu), also showed significant reductions in chlorophyll content and yield-related traits. However, TaCHLI-7D overexpression in rice significantly decreased thousand kernel weight, yield per plant, and germination. Additionally, natural variations in TaCHLI-7A/B are significantly associated with flag leaf, spike exsertion length, and yield per plant. Notably, the favorable haplotype, TaCHLI-7B-HapII, which displayed higher thousand kernel weight and yield per plant, is positively selected in wheat breeding. Our study provides insights on the regulatory molecular mechanisms underpinning leaf color and chlorophyll biosynthesis, and highlights TaCHLI functions, which provide useful molecular markers and genetic resources for wheat breeding.