Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.

CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

Base Editing Landscape Extends to Perform Transversion Mutation.

Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.

CRISPR-Cas9 mediated OsMIR168a knockout reveals its pleiotropy in rice.

MicroRNA168 (MIR168) is a key miRNA that targets the main RNA-induced silencing complex component Argonaute 1 (AGO1) to regulate plant growth and environmental stress responses. However, the regulatory functions of MIR168 need to be further elucidated in rice. In this paper, we generated clean OsMIR168a deletion mutants by CRISPR-Cas9 strategy. We then phenotypically and molecularly characterized these mutants. The rice OsMIR168a mutants grew rapidly at the seedling stage, produced more tillers and matured early. Compared to the wild-type plants, the mutants were shorter at maturity and produced smaller spikelets and seeds. Analysis of gene expression showed that the transcription levels of OsMIR168a's target genes such as OsAGO1a, OsAGO1b and OsAGO1d were elevated significantly in the OsMIR168a mutants. Intriguingly, OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of OsMIR168a not only by informatic prediction but also by expression analysis and a cell-based cleavage assay in the OsMIR168a mutants. Many protein-coding genes and miRNAs showed differential expression in the OsMIR168a mutants, suggesting OsMIR168a exerts a major transcriptional regulatory role, likely through its potential target genes such as OsAGO1s and OsAGO18. KEGG enrichment analysis of these differentially expressed genes pointed to OsMIR168a's involvement in important processes such as plant hormone signalling transduction and plant-pathogen interaction. These data collectively support that the complex regulation module of OsMIR168a-OsAGO1/OsAGO18-miRNAs-target genes contributes to agronomically important traits, which sheds light on miRNA-mediated crop breeding.

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice.

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

CRISPR-BETS: a base-editing design tool for generating stop codons.

Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.

Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor.

Cytosine base editors (CBEs) are the promising tools for precise genome editing in plants. It is important to investigate potential off-target effects of an efficient CBE at the genome and transcriptome levels in a major crop. Based on comparison of five cytidine deaminases and two different promoters for expressing single-guide RNAs (sgRNAs), we tested a highly efficient A3A/Y130F-BE3 system for efficient C-to-T base editing in tomato (Solanum lycopersicum). We then conducted whole-genome sequencing of four base-edited tomato plants, three Green fluorescent protein (GFP)-expressing control plants, and two wild-type plants. The sequencing depths ranged from 25× to 49× with read mapping rates >97%. No sgRNA-dependent off-target mutations were detected. Our data show an average of approximately 1,000 single-nucleotide variations (SNVs) and approximately 100 insertions and deletions (indels) per GFP control plant. Base-edited plants had on average elevated levels of SNVs (approximately 1,250) and indels (approximately 300) per plant. On average, about 200 more C-to-T (G-to-A) mutations were found in a base-edited plant than a GFP control plant, suggesting some level of sgRNA-independent off-target effects, though the difference is not statistically significant. We also conducted RNA sequencing of the same four base-edited plants and three GFP control plants. An average of approximately 200 RNA SNVs was discovered per plant for either base-edited or GFP control plants. Furthermore, no specific enrichment of C-to-U mutations can be found in the base-edited plants. Hence, we cannot find any evidence for bona fide off-target mutations by A3A/Y130F-BE3 at the transcriptome level.

Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice.

CRISPR-Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non-coding RNAs in plants is still at its infancy. Despite being a major class of non-coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR-Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR-Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs' participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA-miRNA-mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.

Improved plant cytosine base editors with high editing activity, purity, and specificity.

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3-like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at A-rich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.

BACKGROUND: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. RESULTS: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. CONCLUSION: Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.

Low pH effects on reactive oxygen species and methylglyoxal metabolisms in Citrus roots and leaves.

BACKGROUND: Limited data are available on the responses of reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms to low pH in roots and leaves. In China, quite a few of Citrus are cultivated in acidic soils (pH < 5.0). 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) (C. sinensis were more tolerant to low pH than C. grandis) seedlings were irrigated daily with nutrient solution at a pH of 2.5, 3 or 5 for nine months. Thereafter, we examined low pH effects on growth, and superoxide anion production rate (SAP), malondialdehyde (MDA), MG, antioxidants, and enzymes related to ROS and MG detoxification in roots and leaves in order to (a) test the hypothesis that low pH affected ROS and MG metabolisms more in roots than those of leaves, and (b) understand the roles of ROS and MG metabolisms in Citrus low pH-tolerance and -toxicity. RESULTS: Compared with control, most of the physiological parameters related to ROS and MG metabolisms were greatly altered at pH 2.5, but almost unaffected at pH 3. In addition to decreased root growth, many fibrous roots became rotten and died at pH 2.5. pH 2.5-induced changes in SAP, the levels of MDA, MG and antioxidants, and the activities of most enzymes related to ROS and MG metabolisms were greater in roots than those of leaves. Impairment of root ascorbate metabolism was the most serious, especially in C. grandis roots. pH 2.5-induced increases in MDA and MG levels in roots and leaves, decreases in the ratios of ascorbate/(ascorbate+dehydroascorbate) in roots and leaves and of reduced glutathione/(reduced+oxidized glutathione) in roots were greater in C. grandis than those in C. sinensis. CONCLUSIONS: Low pH affected MG and ROS metabolisms more in roots than those in leaves. The most seriously impaired ascorbate metabolism in roots was suggested to play a role in low pH-induced root death and growth inhibition. Low pH-treated C. sinensis roots and leaves had higher capacity to maintain a balance between ROS and MG production and their removal via detoxification systems than low pH-treated C. grandis ones, thus contribute to the higher acid-tolerance of C. sinensis.

Responses of reactive oxygen species and methylglyoxal metabolisms to magnesium-deficiency differ greatly among the roots, upper and lower leaves of Citrus sinensis.

BACKGROUND: Magnesium (Mg)-deficiency is one of the most prevalent physiological disorders causing a reduction in Citrus yield and quality. 'Xuegan' (Citrus sinensis) seedlings were irrigated for 16 weeks with nutrient solution containing 2 mM (Mg-sufficiency) or 0 mM (Mg-deficiency) Mg(NO3)2. Thereafter, we investigated the Mg-deficient effects on gas exchange and chlorophyll a fluorescence in the upper and lower leaves, and Mg, reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms in the roots, lower and upper leaves. The specific objectives were to corroborate the hypothesis that the responses of ROS and MG metabolisms to Mg-deficiency were greater in the lower leaves than those in the upper leaves, and different between the leaves and roots. RESULTS: Mg level was higher in the Mg-deficient upper leaves than that in the Mg-deficient lower leaves. This might be responsible for the Mg-deficiency-induced larger alterations of all the measured parameters in the lower leaves than those in the upper leaves, but they showed similar change patterns between the Mg-deficient lower and upper leaves. Accordingly, Mg-deficiency increased greatly their differences between the lower and upper leaves. Most of parameters involved in ROS and MG metabolisms had similar variation trends and degrees between the Mg-deficient lower leaves and roots, but several parameters (namely glutathione S-transferase, sulfite reductase, ascorbate and dehydroascorbate) displayed the opposite variation trends. Obviously, differences existed in the Mg-deficiency-induced alterations of ROS and MG metabolisms between the lower leaves and roots. Although the activities of most antioxidant and sulfur metabolism-related enzymes and glyoxalase I and the level of reduced glutathione in the Mg-deficient leaves and roots and the level of ascorbate in the leaves were kept in higher levels, the levels of malonaldehyde and MG and/or electrolyte leakage were increased in the Mg-deficient lower and upper leaves and roots, especially in the Mg-deficient lower leaves and roots. CONCLUSIONS: The ROS and MG detoxification systems as a whole did not provide sufficient detoxification capacity to prevent the Mg-deficiency-induced production and accumulation of ROS and MG, thus leading to lipid peroxidation and the loss of plasma membrane integrity, especially in the lower leaves and roots.

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize.

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing.

CRISPR-Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species-specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first-generation single transcript unit (STU)-Cas9 system, STU-Cas9-RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU-Cas9 systems and one STU-Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU-Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU-Cas9-Csy4 and STU-Cas9-tRNA systems showed more robust genome editing efficiencies than our first-generation STU-Cas9-RZ system and the conventional mixed dual promoter system. We further applied the STU-Cas9-tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU-based PmCDA1 base editor system is highly efficient in rice. The STU-Cas12a system, based on Cas12a' self-processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

Multiplex QTL editing of grain-related genes improves yield in elite rice varieties.

KEY MESSAGE: Significant yield increase has been achieved by simultaneous introduction of three trait-related QTLs in three rice varieties with multiplex editing by CRISPR-Cas9. Using traditional breeding approaches to develop new elite rice varieties with high yield and superior quality is challenging. It usually requires introduction of multiple trait-related quantitative trait loci (QTLs) into an elite background through multiple rounds of crossing and selection. CRISPR-Cas9-based multiplex editing of QTLs represents a new breeding strategy that is straightforward and cost effective. To test this approach, we simultaneously targeted three yield-related QTLs for editing in three elite rice varieties, namely J809, L237 and CNXJ. The chosen yield-related QTL genes are OsGS3, OsGW2 and OsGn1a, which have been identified to negatively regulate the grain size, width and weight, and number, respectively. Our approach rapidly generated all seven combinations of single, double and triple mutants for the target genes in elite backgrounds. Detailed analysis of these mutants revealed differential contributions of QTL mutations to yield performance such as grain length, width, number and 1000-grain weight. Overall, the contributions are additive, resulting in 68 and 30% yield per panicle increase in triple mutants of J809 and L237, respectively. Our data hence demonstrates a promising genome editing approach for rapid breeding of QTLs in elite crop varieties.

Low pH-responsive proteins revealed by a 2-DE based MS approach and related physiological responses in Citrus leaves.

BACKGROUND: Rare data are available on the molecular responses of higher plants to low pH. Seedlings of 'Sour pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were treated daily with nutrient solution at a pH of 2.5, 3, or 6 (control) for nine months. Thereafter, we first used 2-dimensional electrophoresis (2-DE) to investigate low pH-responsive proteins in Citrus leaves. Meanwhile, we examined low pH-effects on leaf gas exchange, carbohydrates, ascorbate, dehydroascorbate and malondialdehyde. The objectives were to understand the adaptive mechanisms of Citrus to low pH and to identify the possible candidate proteins for low pH-tolerance. RESULTS: Our results demonstrated that Citrus were tolerant to low pH, with a slightly higher low pH-tolerance in the C. sinensis than in the C. grandis. Using 2-DE, we identified more pH 2.5-responsive proteins than pH 3-responsive proteins in leaves. This paper discussed mainly on the pH 2.5-responsive proteins. pH 2.5 decreased the abundances of proteins involved in ribulose bisphosphate carboxylase/oxygenase activation, Calvin cycle, carbon fixation, chlorophyll biosynthesis and electron transport, hence lowering chlorophyll level, electron transport rate and photosynthesis. The higher oxidative damage in the pH 2.5-treated C. grandis leaves might be due to a combination of factors including higher production of reactive oxygen species, more proteins decreased in abundance involved in antioxidation and detoxification, and lower ascorbate level. Protein and amino acid metabolisms were less affected in the C. sinensis leaves than those in the C. grandis leaves when exposed to pH 2.5. The abundances of proteins related to jasmonic acid biosynthesis and signal transduction were increased and decreased in the pH 2.5-treated C. sinensis and C. grandis leaves, respectively. CONCLUSIONS: This is the first report on low pH-responsive proteins in higher plants. Thus, our results provide some novel information on low pH-toxicity and -tolerance in higher plants.