Molecular Mechanisms of Plant Responses to Copper: From Deficiency to Excess.

Copper (Cu) is an essential nutrient for plant growth and development. This metal serves as a constituent element or enzyme cofactor that participates in many biochemical pathways and plays a key role in photosynthesis, respiration, ethylene sensing, and antioxidant systems. The physiological significance of Cu uptake and compartmentalization in plants has been underestimated, despite the importance of Cu in cellular metabolic processes. As a micronutrient, Cu has low cellular requirements in plants. However, its bioavailability may be significantly reduced in alkaline or organic matter-rich soils. Cu deficiency is a severe and widespread nutritional disorder that affects plants. In contrast, excessive levels of available Cu in soil can inhibit plant photosynthesis and induce cellular oxidative stress. This can affect plant productivity and potentially pose serious health risks to humans via bioaccumulation in the food chain. Plants have evolved mechanisms to strictly regulate Cu uptake, transport, and cellular homeostasis during long-term environmental adaptation. This review provides a comprehensive overview of the diverse functions of Cu chelators, chaperones, and transporters involved in Cu homeostasis and their regulatory mechanisms in plant responses to varying Cu availability conditions. Finally, we identified that future research needs to enhance our understanding of the mechanisms regulating Cu deficiency or stress in plants. This will pave the way for improving the Cu utilization efficiency and/or Cu tolerance of crops grown in alkaline or Cu-contaminated soils.

Identification of qGL4.1 and qGL4.2, two closely linked QTL controlling grain length in rice.

Grain size is an important appearance quality trait in rice, which also affects grain yield. In this study, a recombinant inbred line (RIL) population derived from a cross between indica variety 9311 and japonica variety Cypress was constructed. And 181 out of 600 RILs were sequenced, and a high-density genetic map containing 2842 bin markers was constructed, with a total map length of 1500.6 cM. A total of 10 quantitative trait loci (QTL) related to grain length (GL), grain width (GW), grain length-to-width ratio (LWR), and 1000-grain weight (TGW) were detected under two environments. The genetic effect of qGL4, a minor QTL for GL and TGW, was validated using three heterogeneous inbred family (HIF) segregation populations. It was further dissected into two closed linked QTL, qGL4.1 and qGL4.2. By progeny testing, qGL4.1 and qGL4.2 were successfully delimited to intervals of 1304-kb and 423-kb, respectively. Our results lay the foundation for the map-based cloning of qGL4.1 and qGL4.2 and provide new gene resources for the improvement of grain yield and quality in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01447-y.

CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice.

Amylose content (AC) is one of the physicochemical indexes of rice quality, which is largely determined by the Waxy (Wx) gene. Fragrance in rice is favored because it adds good flavor and a faint scent. Loss of function of the BADH2 (FGR) gene promotes the biosynthesis of 2-acetyl-1-pyrroline (2AP), which is the main compound responsible for aroma in rice. Here, we used a CRISPR/Cas9 system to simultaneously knock out Wx and FGR genes in 1892S and M858, which are the parents of an indica two-line hybrid rice, Huiliangyou 858 (HLY858). Four T-DNA-free homozygous mutants (1892Swxfgr-1, 1892Swxfgr-2, M858wxfgr-1, and M858wxfgr-2) were obtained. The 1892Swxfgr and M858wxfgr were crossed to generate double mutant hybrid lines HLY858wxfgr-1 and HLY858wxfgr-2. Size-exclusion chromatography (SEC) data indicated that true AC of the wx mutant starches ranged from 0.22 to 1.63%, much lower than those of the wild types (12.93 to 13.76%). However, the gelatinization temperature (GT) of the wx mutants in backgrounds of 1892S, M858, and HLY858 were still high, and showed no significant differences with the wild type controls. The aroma compounds 2AP content in grains of HLY858wxfgr-1 and HLY858wxfgr-2 were 153.0 µg/kg and 151.0 µg/kg, respectively. In contrast, 2AP was not detected in grains of HLY858. There were no significant differences in major agronomic traits between the mutants and HLY858. This study provides guidelines for cultivation of ideal glutinous and aromatic hybrid rice by gene editing.

Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice.

BACKGROUND: Ribosomes responsible for transcription and translation of plastid-encoded proteins in chloroplasts are essential for chloroplast development and plant growth. Although most ribosomal proteins in plastids have been identified, the molecular mechanisms regulating chloroplast biogenesis remain to be investigated. RESULTS: Here, we identified albinic seedling mutant albino seedling lethality 4 (asl4) caused by disruption of 30S ribosomal protein S1 that is targeted to the chloroplast. The mutant was defective in early chloroplast development and chlorophyll (Chl) biosynthesis. A 2855-bp deletion in the ASL4 allele was verified as responsible for the mutant phenotype by complementation tests. Expression analysis revealed that the ASL4 allele was highly expressed in leaf 4 sections and newly expanded leaves during early leaf development. Expression levels were increased by exposure to light following darkness. Some genes involved in chloroplast biogenesis were up-regulated and others down-regulated in asl4 mutant tissues compared to wild type. Plastid-encoded plastid RNA polymerase (PEP)-dependent photosynthesis genes and nuclear-encoded phage-type RNA polymerase (NEP)-dependent housekeeping genes were separately down-regulated and up-regulated, suggesting that plastid transcription was impaired in the mutant. Transcriptome and western blot analyses showed that levels of most plastid-encoded genes and proteins were reduced in the mutant. The decreased contents of chloroplast rRNAs and ribosomal proteins indicated that chloroplast ribosome biogenesis was impaired in the asl4 mutant. CONCLUSIONS: Rice ASL4 encodes 30S ribosomal protein S1, which is targeted to the chloroplast. ASL4 is essential for chloroplast ribosome biogenesis and early chloroplast development. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

OPEN GLUME1: a key enzyme reducing the precursor of JA, participates in carbohydrate transport of lodicules during anthesis in rice.

KEY MESSAGE: OG1 is involved in JA-regulated anthesis by modulating carbohydrate transport of lodicules in rice. Flowering plants have evolved a sophisticated regulatory network to coordinate anthesis and maximize reproductive success. In addition to various environmental conditions, the plant hormone jasmonic acid and its derivatives (JAs) are involved in anthesis. However, the underlying mechanism remains largely unexplored. Here, we report a JA-defective mutant in rice (Oryza sativa), namely open glume 1, which has dysfunctional lodicules that lead to open glumes following anthesis. Map-based cloning and subsequent complementation tests confirmed that OG1 encodes a peroxisome-localized 12-oxo-phytodienoic acid reductase-a key enzyme that reduces the precursor of JA. Loss-of-function of OG1 resulted in almost no JA accumulation. Exogenous JA treatment completely rescued the defects caused by the og1 mutation. Further studies revealed that intracellular metabolism was disrupted in the lodicules of og1 mutant. At the mature plant stage, most seeds of the mutant were malformed with significantly reduced starch content. We speculate that JA or JA signaling mediates the carbohydrate transport of lodicules during anthesis, and signal the onset of cell degradation in lodicules after anthesis. We conclude that the OPEN GLUME 1 gene that produces a key enzyme involved in reducing the precursor of JA in JA biosynthesis and is involved in carbohydrate transport underlying normal lodicule function during anthesis in rice.

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis.

Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis

Abstract Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development.

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice.

GW5 acts in the brassinosteroid signalling pathway to regulate grain width and weight in rice.

Grain size is a major determinant of grain yield in cereal crops. qSW5/GW5, which exerts the greatest effect on rice grain width and weight, was fine-mapped to a 2,263-bp/21-kb genomic region containing a 1,212-bp deletion, respectively. Here, we show that a gene encoding a calmodulin binding protein, located ∼5 kb downstream of the 1,212-bp deletion, corresponds to qSW5/GW5. GW5 is expressed in various rice organs, with highest expression level detected in young panicles. We provide evidence that the 1,212-bp deletion affects grain width most likely through influencing the expression levels of GW5. GW5 protein is localized to the plasma membrane and can physically interact with and repress the kinase activity of rice GSK2 (glycogen synthase kinase 2), a homologue of Arabidopsis BIN2 (BRASSINOSTEROID INSENSITIVE2) kinase, resulting in accumulation of unphosphorylated OsBZR1 (Oryza sativa BRASSINAZOLE RESISTANT1) and DLT (DWARF AND LOW-TILLERING) proteins in the nucleus to mediate brassinosteroid (BR)-responsive gene expression and growth responses (including grain width and weight). Our results suggest that GW5 is a novel positive regulator of BR signalling and a viable target for genetic manipulation to improve grain yield in rice and perhaps in other cereal crops as well.

The LBD12-1 Transcription Factor Suppresses Apical Meristem Size by Repressing Argonaute 10 Expression.

The shoot apical meristem (SAM) consists of a population of multipotent cells that generates all aerial structures and regenerates itself. SAM maintenance and lateral organ development are regulated by several complex signaling pathways, in which the Argonaute gene-mediated pathway plays a key role. One Argonaute gene, AGO10, functions as a microRNA locker that attenuates miR165/166 activity and positively regulates shoot apical meristem development, but little is known about when and how AGO10 is regulated at the transcriptional level. In this work, we showed that transgenic rice plants overexpressing LBD12-1, an LBD family transcription factor, exhibited stunted growth, twisted leaves, abnormal anthers, and reduced SAM size. Further research revealed that LBD12-1 directly binds to the promoter region and represses the expression of AGO10. Overexpression of AGO10 in an LBD12-1 overexpression background rescued the growth defect phenotype of LBD12-1-overexpressing plants. The expression of LBD12-1 and its binding ability to the AGO10 promoter is induced by stress. lbd12-1 loss-of-function mutants showed similar phenotypes and SAM size to the wild type under normal conditions, but lbd12-1 had a larger SAM under salt stress. Our findings provide novel insights into the regulatory mechanism of AGO10 by which SAM size is controlled under stress conditions.

Young Seedling Stripe1 encodes a chloroplast nucleoid-associated protein required for chloroplast development in rice seedlings.

MAIN CONCLUSION: Young Seedling Stripe1 (YSS1) was characterized as an important regulator of plastid-encoded plastid RNA polymerase (PEP) activity essential for chloroplast development at rice seedling stage. Chloroplast development is coordinately regulated by plastid- and nuclear-encoding genes. Although a few regulators have been reported to be involved in chloroplast development, new factors remain to be identified, given the complexity of this process. Here, we report the characterization of a temperature-sensitive young seedling stripe1 (yss1) rice mutant, which develops striated leaves at the seedling stage, particularly in leaf 3, but produces wild-type leaves in leaf 5 and onwards. The chlorotic leaves have decreased chlorophyll (Chls) accumulation and impaired chloroplast structure. Positional cloning combined with sequencing demonstrated that aberrant splicing of the 8th intron in YSS1 gene, due to a single nucleotide deletion around splicing donor site, leads to decreased expression of YSS1 and accumulation of an 8th intron-retained yss1 transcript. Furthermore, complementation test revealed that downregulation of YSS1 but not accumulation of yss1 transcript confers yss1 mutant phenotype. YSS1 encodes a chloroplast nucleoid-localized protein belonging to the DUF3727 superfamily. Expression analysis showed that YSS1 gene is more expressed in newly expanded leaves, and distinctly up-regulated as temperatures increase and by light stimulus. PEP- and nuclear-encoded phage-type RNA polymerase (NEP)-dependent genes are separately down-regulated and up-regulated in yss1 mutant, indicating that PEP activity may be impaired. Furthermore, levels of chloroplast proteins are mostly reduced in yss1 seedlings. Together, our findings identify YSS1 as a novel regulator of PEP activity essential for chloroplast development at rice seedling stage.

WSL3, a component of the plastid-encoded plastid RNA polymerase, is essential for early chloroplast development in rice.

Plastid-encoded plastid RNA polymerase (PEP), a dominant RNA polymerase in mature chloroplasts, consists of core subunits and peripheral subunits. Despite the importance of the peripheral subunits in control of PEP activity it is unclear how they interact with one another to exert physiological effects on chloroplast development and plant growth, especially in rice. Here, we report a mutant, designated wsl3 that lacks a peripheral subunit in rice. We isolated the WSL3 gene encoding an essential peripheral subunit of rice PEP complex, OsPAP1/OspTAC3 by map-based cloning, and verified its function by complementation analysis. The wsl3 mutant showed a typical expression pattern of plastid-encoded genes, suggesting that PEP activity was impaired. Using immunofluorescent labeling and immunoblotting, we found that WSL3 was localized to the chloroplast and associated with the nucleoid. In addition, we demonstrated that WSL3 interacted with PEP subunits in Y2H, BiFC and pull-down experiments. Furthermore, a cpDNA IP assay revealed that WSL3 was associated with the PEP complex during the entire transcription process. We provide evidence suggesting that WSL3 is essential for early chloroplast development by interacting with subunits of the PEP complex.

Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis.

The uppermost internode is one of the fastest elongating organs in rice, and is expected to require an adequate supply of cell-wall materials and enzymes to the cell surface to enhance mechanical strength. Although it has been reported that the phenotype of shortened uppermost internode 1 (sui1) is caused by mutations in PHOSPHATIDYLSERINE SYNTHASE (OsPSS), the underlying mechanism remains unclear. Here we show that the OsPSS-1, as a gene expressed predominantly in elongating cells, regulates post-Golgi vesicle secretion to intercellular spaces. Mutation of OsPSS-1 leads to compromised delivery of CESA4 and secGFP towards the cell surface, resulting in weakened intercellular adhesion and disorganized cell arrangement in parenchyma. The phenotype of sui1-4 is caused largely by the reduction in cellulose contents in the whole plant and detrimental delivery of pectins in the uppermost internode. We found that OsPSS-1 and its potential product PS (phosphatidylserine) localized to organelles associated with exocytosis. These results together suggest that OsPSS-1 plays a potential role in mediating cell expansion by regulating secretion of cell wall components.

BRITTLE SHEATH1 encoding OsCYP96B4 is involved in secondary cell wall formation in rice.

KEY MESSAGE: Mutation of BSH1 leads to brittle sheath phenotype and reduction of very-long-chain fatty acids and their derivatives in wax. The cell wall plays an important role in plant mechanical strength. Several brittle culm mutants have been identified and characterized in rice. Here, we characterized an anther culture-derived rice brittle sheath mutant, named bsh1 and isolated BSH1 via map-based strategy. BSH1 encodes OsCYP96B4 protein, which was localized on ER membrane in the protoplast transient assay. BSH1 is mainly expressed in developing vascular tissues and the cells in which cell wall secondary thickening is occurring. Mutation in bsh1 causes changes in cell wall composition by affecting the expression of cell wall-related genes. Moreover, bsh1 shows reduced amounts of very-long-chain fatty acids and their derivatives in wax rather than the medium-chain fatty acids. In summary, BSH1 functions mainly in secondary cell wall formation, and probably in wax biosynthesis in an unidentified mechanism.

An evolutionarily conserved gene, FUWA, plays a role in determining panicle architecture, grain shape and grain weight in rice.

Plant breeding relies on creation of novel allelic combinations for desired traits. Identification and utilization of beneficial alleles, rare alleles and evolutionarily conserved genes in the germplasm (referred to as 'hidden' genes) provide an effective approach to achieve this goal. Here we show that a chemically induced null mutation in an evolutionarily conserved gene, FUWA, alters multiple important agronomic traits in rice, including panicle architecture, grain shape and grain weight. FUWA encodes an NHL domain-containing protein, with preferential expression in the root meristem, shoot apical meristem and inflorescences, where it restricts excessive cell division. Sequence analysis revealed that FUWA has undergone a bottleneck effect, and become fixed in landraces and modern cultivars during domestication and breeding. We further confirm a highly conserved role of FUWA homologs in determining panicle architecture and grain development in rice, maize and sorghum through genetic transformation. Strikingly, knockdown of the FUWA transcription level by RNA interference results in an erect panicle and increased grain size in both indica and japonica genetic backgrounds. This study illustrates an approach to create new germplasm with improved agronomic traits for crop breeding by tapping into evolutionary conserved genes.

Dwarf and tiller-enhancing 1 regulates growth and development by influencing boron uptake in boron limited conditions in rice.

Boron (B) is essential for plant growth, and B deficiency causes severe losses in crop yield. Here we isolated and characterized a rice (Oryza sativa L.) mutant named dwarf and tiller-enhancing 1 (dte1), which exhibits defects under low-B conditions, including retarded growth, increased number of tillers and impaired pollen fertility. Map-based cloning revealed that dte1 encodes a NOD26-LIKE INTRINSIC PROTEIN orthologous to known B channel proteins AtNIP5;1 in Arabidopsis and TASSEL-LESS1 in maize. Its identity was verified by transgenic complementation and RNA-interference. Subcellular localization showed DTE1 is mainly localized in the plasma membrane. The accumulation of DTE1 transcripts both in roots and shoots significantly increased within 3h of the onset of B starvation, but decreased within 1h of B replenishment. GUS staining indicated that DTE1s are expressed abundantly in exodermal cells in roots, as well as in nodal region of adult leaves. Although the dte1 mutation apparently reduces the total B content in plants, it does not affect in vivo B concentrations under B-deficient conditions. These data provide evidence that DTE1 is critical for vegetative growth and reproductive development in rice grown under B-deficient conditions.

Pi64, Encoding a Novel CC-NBS-LRR Protein, Confers Resistance to Leaf and Neck Blast in Rice.

Rice blast caused by Magnaporthe oryzae poses a major threat to rice production worldwide. The utilization of host resistance (R) genes is considered to be the most effective and economic means to control rice blast. Here, we show that the japonica landrace Yangmaogu (YMG) displays a broader spectrum of resistance to blast isolates than other previously reported broad-spectrum resistant (BSR) cultivars. Genetic analysis suggested that YMG contains at least three major R genes. One gene, Pi64, which exhibits resistance to indica-sourced isolate CH43 and several other isolates, was mapped to a 43-kb interval on chromosome 1 of YMG. Two open reading frames (NBS-1 and NBS-2) encoding nucleotide-binding site and leucine-rich repeat proteins were short-listed as candidate genes for Pi64. Constructs containing each candidate gene were transformed into three susceptible japonica cultivars. Only transformants with NBS-2 conferred resistance to leaf and neck blast, validating the idea that NBS-2 represents the functional Pi64 gene. Pi64 is constitutively expressed at all development stages and in all tissues examined. Pi64 protein is localized in both the cytoplasm and nucleus. Furthermore, introgression of Pi64 into susceptible cultivars via gene transformation and marker-assisted selection conferred high-level and broad-spectrum leaf and neck blast resistance to indica-sourced isolates, demonstrating its potential utility in breeding BSR rice cultivars.