An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants.

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Chromosome-scale assembly and gene editing of Solanum americanum genome reveals the basis for thermotolerance and fruit anthocyanin composition.

Solanum americanum serves as a promising source of resistance genes against potato late blight and is considered as a leafy vegetable for complementary food and nutrition. The limited availability of high-quality genome assemblies and gene annotations has hindered the exploration and exploitation of stress-resistance genes in S. americanum. Here, we present a chromosome-level genome assembly of a thermotolerant S. americanum ecotype and identify a crucial heat-inducible transcription factor gene, SaHSF17, essential for heat tolerance. The CRISPR/Cas9 system-mediated knockout of SaHSF17 results in remarkably reduced thermotolerance in S. americanum, exhibiting a significant suppression of multiple HSP gene expressions under heat treatment. Furthermore, our transcriptome analysis and anthocyanin component investigation of fruits indicated that delphinidins are the major anthocyanins accumulated in the mature dark-purple fruits. The accumulation of delphinidins and other pigment components during fruit ripening in S. americanum coincides with the transcriptional regulation of key genes, particularly the F3'5'H and F3'H genes, in the anthocyanin biosynthesis pathway. By integrating existing knowledge, the development of this high-quality reference genome for S. americanum will facilitate the identification and utilization of novel abiotic and biotic stress-resistance genes for improvement of Solanaceae and other crops.

APETALA2 is involved in ABA signaling during seed germination.

APETALA2 (AP2) is well known for regulating the development of floral organs, ovules, seed coats, and the mass of seeds, but the role of AP2 in seed germination remains unclear. Here, we report that AP2 interacts with ABI5 in nuclear speckles and functions in controlling seed germination. Genetic study showed that the abi5 mutation could restore the ABA-sensitive phenotype of ap2 mutants, supporting that AP2 antagonizes ABI5 in ABA signaling and ABA-mediated inhibition of seed germination. In addition, we observed the interactions of AP2 with SnRK2.2, SnRK2.3, and SnRK2.6 in nuclear speckles, suggesting that AP2 plays a multifaceted role in the ABA signaling pathway. Our findings revealed that the interactions of AP2 with SnRK2s and ABI5 are critical for ABA signaling in control of seed germination.

Natural variation in SlSOS2 promoter hinders salt resistance during tomato domestication.

Increasing soil salinization seriously impairs plant growth and development, resulting in crop loss. The Salt-Overly-Sensitive (SOS) pathway is indispensable to the mitigation of Na + toxicity in plants under high salinity. However, whether natural variations of SOS2 contribute to salt tolerance has not been reported. Here a natural variation in the SlSOS2 promoter region was identified to be associated with root Na+/K+ ratio and the loss of salt resistance during tomato domestication. This natural variation contains an ABI4-binding cis-element and plays an important role in the repression of SlSOS2 expression. Genetic evidence revealed that SlSOS2 mutations increase root Na+/K+ ratio under salt stress conditions and thus attenuate salt resistance in tomato. Together, our findings uncovered a critical but previously unknown natural variation of SOS2 in salt resistance, which provides valuable natural resources for genetic breeding for salt resistance in cultivated tomatoes and other crops.

The Sm core protein SmEb regulates salt stress responses through maintaining proper splicing of RCD1 pre-mRNA in Arabidopsis.

Salt stress adversely impacts crop production. Several spliceosome components have been implicated in regulating salt stress responses in plants, however, the underlying molecular basis is still unclear. Here we report that the spliceosomal core protein SmEb is essential to salt tolerance in Arabidopsis. Transcriptome analysis showed that SmEb modulates alternative splicing of hundreds of pre-mRNAs in plant response to salt stress. Further study revealed that SmEb is crucial in maintaining proper ratio of two RCD1 splicing variants (RCD1.1/RCD1.2) important for salt stress response. In addition, RCD1.1 but not RCD1.2 is able to interact with the stress regulators and attenuates salt-sensitivity by decreasing salt-induced cell death in smeb-1 mutant. Together, our findings uncovered the essential role of SmEb in the regulation of alternative pre-mRNA splicing in salt stress response.

Modulation of plant development and chilling stress responses by alternative splicing events under control of the spliceosome protein SmEb in Arabidopsis.

Cold stress resulting from chilling and freezing temperatures substantially inhibits plant growth and reduces crop production worldwide. Tremendous research efforts have been focused on elucidating the molecular mechanisms of freezing tolerance in plants. However, little is known about the molecular nature of chilling stress responses in plants. Here we found that two allelic mutants in a spliceosome component gene SmEb (smeb-1 and smeb-2) are defective in development and responses to chilling stress. RNA-seq analysis revealed that SmEb controls the splicing of many pre-messenger RNAs (mRNAs) under chilling stress. Our results suggest that SmEb is important to maintain proper ratio of the two COP1 splicing variants (COP1a/COP1b) to fine tune the level of HY5. In addition, the transcription factor BES1 shows a dramatic defect in pre-mRNA splicing in the smeb mutants. Ectopic expression of the two BES1 splicing variants enhances the chilling sensitivity of the smeb-1 mutant. Furthermore, biochemical and genetic analysis showed that CBFs act as negative upstream regulators of SmEb by directly suppressing its transcription. Together, our results demonstrate that proper alternative splicing of pre-mRNAs controlled by the spliceosome component SmEb is critical for plant development and chilling stress responses.

Transcriptomic and metabolomic landscape of quinoa during seed germination.

BACKGROUND: Quinoa (Chenopodium quinoa), a dicotyledonous species native to Andean region, is an emerging crop worldwide nowadays due to its high nutritional value and resistance to extreme abiotic stresses. Although it is well known that seed germination is an important and multiple physiological process, the network regulation of quinoa seed germination is largely unknown. RESULTS: Here, we performed transcriptomic study in five stages during transition from quinoa dry seed to seedling. Together with the GC-MS based metabolome analysis, we found that seed metabolism is reprogrammed with significant alteration of multiple phytohormones (especially abscisic acid) and other nutrients during the elongation of radicels. Cell-wall remodeling is another main active process happening in the early period of quinoa seed germination. Photosynthesis was fully activated at the final stage, promoting the biosynthesis of amino acids and protein to allow seedling growth. The multi-omics analysis revealed global changes in metabolic pathways and phenotype during quinoa seed germination. CONCLUSION: The transcriptomic and metabolomic landscape depicted here pave ways for further gene function elucidation and quinoa development in the future.

SUMO E3 ligase SIZ1 negatively regulates arsenite resistance via depressing GSH biosynthesis in Arabidopsis.

Arsenic is a metalloid toxic to plants, animals and human beings. Small ubiquitin-like modifier (SUMO) conjugation is involved in many biological processes in plants. However, the role of SUMOylation in regulating plant arsenic response is still unclear. In this study, we found that dysfunction of SUMO E3 ligase SIZ1 improves arsenite resistance in Arabidopsis. Overexpression of the dominant-negative SUMO E2 variant resembled the arsenite-resistant phenotype of siz1 mutant, indicating that SUMOylation plays a negative role in plant arsenite detoxification. The siz1 mutant accumulated more glutathione (GSH) than the wild type under arsenite stress, and the arsenite-resistant phenotype of siz1 was depressed by inhibiting GSH biosynthesis. The transcript levels of the genes in the GSH biosynthetic pathway were increased in the siz1 mutant comparing with the wild type in response to arsenite treatment. Taken together, our findings revealed a novel function of SIZ1 in modulating plant arsenite response through regulating the GSH-dependent detoxification.

Crafting the plant root metabolome for improved microbe-assisted stress resilience.

Plants and microbes coinhabit the earth and have coevolved during environmental changes over time. Root metabolites are the key to mediating the dynamic association between plants and microbes, yet the underlying functions and mechanisms behind this remain largely illusive. Knowledge of metabolite-mediated alteration of the root microbiota in response to environmental stress will open avenues for engineering root microbiotas for improved plant stress resistance and health. Here, we synthesize recent advances connecting environmental stresses, the root metabolome and microbiota, and propose integrated synthetic biology-based strategies for tuning the plant root metabolome in situ for microbe-assisted stress resistance, offering potential solutions to combat climate change. The current limitations, challenges and perspectives for engineering the plant root metabolome for modulating microbiota are collectively discussed.

The Arabidopsis spliceosomal protein SmEb modulates ABA responses by maintaining proper alternative splicing of HAB1.

Abscisic acid (ABA) signaling is critical for seed germination and abiotic stress responses in terrestrial plants. Pre-mRNA splicing is known to regulate ABA signaling. However, the involvement of canonical spliceosomal components in regulating ABA signaling is poorly understood. Here, we show that the spliceosome component Sm core protein SmEb plays an important role in ABA signaling. SmEb expression is up-regulated by ABA treatment, and analysis of Arabidopsis smeb mutant plants suggest that SmEb modulates the alternative splicing of the ABA signaling component HAB1 by enhancing the HAB1.1 splicing variant while repressing HAB1.2. Overexpression of HAB1.1 but not HAB1.2 rescues the ABA-hypersensitive phenotype of smeb mutants. Mutations in the transcription factor ABI3, 4, or 5 also reduce the ABA hypersensitivity of smeb mutants during seed germination. Our results show that the spliceosomal component SmEb plays an important role in ABA regulation of seed germination and early seedling development.

SIZ1-Mediated SUMOylation of ROS1 Enhances Its Stability and Positively Regulates Active DNA Demethylation in Arabidopsis.

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hyper-methylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately 1000 genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared with that in wild-type plants, the protein level of ROS1 is significantly decreased, whereas there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1-mediated SUMOylation of ROS1 promotes its stability and positively regulates active DNA demethylation.

Reciprocal regulation between nicotinamide adenine dinucleotide metabolism and abscisic acid and stress response pathways in Arabidopsis.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living organisms. The homeostasis of NAD is required for plant growth, development, and adaption to environmental cues. In this study, we isolated a chilling hypersensitive Arabidopsis thaliana mutant named qs-2 and identified the causal mutation in the gene encoding quinolinate synthase (QS) critical for NAD biosynthesis. The qs-2 mutant is also hypersensitive to salt stress and abscisic acid (ABA) but resistant to drought stress. The qs-2 mutant accumulates a reduced level of NAD and over-accumulates reactive oxygen species (ROS). The ABA-hypersensitivity of qs-2 can be rescued by supplementation of NAD precursors and by mutations in the ABA signaling components SnRK2s or RBOHF. Furthermore, ABA-induced over-accumulation of ROS in the qs-2 mutant is dependent on the SnRK2s and RBOHF. The expression of QS gene is repressed directly by ABI4, a transcription factor in the ABA response pathway. Together, our findings reveal an unexpected interplay between NAD biosynthesis and ABA and stress signaling, which is critical for our understanding of the regulation of plant growth and stress responses.

Loss of salt tolerance during tomato domestication conferred by variation in a Na+ /K+ transporter.

Domestication has resulted in reduced salt tolerance in tomato. To identify the genetic components causing this deficiency, we performed a genome-wide association study (GWAS) for root Na+ /K+ ratio in a population consisting of 369 tomato accessions with large natural variations. The most significant variations associated with root Na+ /K+ ratio were identified within the gene SlHAK20 encoding a member of the clade IV HAK/KUP/KT transporters. We further found that SlHAK20 transports Na+ and K+ and regulates Na+ and K+ homeostasis under salt stress conditions. A variation in the coding sequence of SlHAK20 was found to be the causative variant associated with Na+ /K+ ratio and confer salt tolerance in tomato. Knockout mutations in tomato SlHAK20 and the rice homologous genes resulted in hypersensitivity to salt stress. Together, our study uncovered a previously unknown molecular mechanism of salt tolerance responsible for the deficiency in salt tolerance in cultivated tomato varieties. Our findings provide critical information for molecular breeding to improve salt tolerance in tomato and other crops.

Two Chloroplast Proteins Negatively Regulate Plant Drought Resistance Through Separate Pathways.

Drought is one of the most deleterious environmental conditions affecting crop growth and productivity. Here we report the important roles of a nuclear-encoded chloroplast protein, PsbP Domain Protein 5 (PPD5), in drought resistance in Arabidopsis (Arabidopsis thaliana). From a forward genetic screen, a drought-resistant mutant named ppd5-2 was identified, which has a knockout mutation in PPD5 The ppd5 mutants showed increased H2O2 accumulation in guard cells and enhanced stomatal closure in response to drought stress. Further analysis revealed that the chloroplast-localized PPD5 protein interacts with and is phosphorylated by OST1, and phosphorylation of PPD5 increases its protein stability. Double mutant ppd5-2ost1-3 exhibited phenotypes resembling the ost1-3 single mutant with decreased stomatal closure, increased water loss, reduced H2O2 accumulation in guard cells, and hypersensitivity to drought stress. These results indicate that the chloroplast protein PPD5 negatively regulates drought resistance by modulating guard cell H2O2 accumulation via an OST1-dependent pathway. Interestingly, the thf1-1 mutant defective in the chloroplast protein THF1 displayed drought-resistance and H2O2 accumulation similar to the ppd5 mutants, but the thf1-1ost1-3 double mutant resembled the phenotypes of the thf1-1 single mutant. These results indicate that both OST1-dependent and OST1-independent pathways exist in the regulation of H2O2 production in chloroplasts of guard cells under drought stress conditions. Additionally, our findings suggest a strategy to improve plant drought resistance through manipulation of chloroplast proteins.

A Role for PICKLE in the Regulation of Cold and Salt Stress Tolerance in Arabidopsis.

Arabidopsis PICKLE (PKL) is a putative CHD3-type chromatin remodeling factor with important roles in regulating plant growth and development as well as RNA-directed DNA methylation (RdDM). The role of PKL protein in plant abiotic stress response is still poorly understood. Here, we report that PKL is important for cold stress response in Arabidopsis. Loss-of-function mutations in the PKL gene lead to a chlorotic phenotype in seedlings under cold stress, which is caused by the alterations in the transcript levels of some chlorophyll metabolism-related genes. The pkl mutant also exhibits increased electrolyte leakage after freezing treatment. These results suggest that PKL is required for proper chilling and freezing tolerance in plants. Gene expression analysis shows that CBF3, encoding a key transcription factor involved in the regulation of cold-responsive genes, exhibits an altered transcript level in the pkl mutant under cold stress. Transcriptome data also show that PKL regulates the expression of a number of cold-responsive genes, including RD29A, COR15A, and COR15B, possibly through its effect on the expression of CBF3 gene. Mutation in PKL gene also results in decreased cotyledon greening rate and reduced primary root elongation under high salinity. Together, our results suggest that PKL regulates plant responses to cold and salt stress.

Structure determination and activity manipulation of the turfgrass ABA receptor FePYR1.

Turfgrass are widely cultivated ornamental plants that have important ecological, societal and economical values. However, many turfgrass species are susceptible to drought and demand frequent irrigation thus consuming large amounts of water. With the ultimate goal of improving drought resistance in turfgrass, we identified several ABA receptors in turfgrass that are important to mediate ABA signaling and drought stress response. The ABA receptor FePYR1 from turfgrass Festuca elata was demonstrated to bind ABA as a monomer. Crystal structure analysis revealed that FePYR1 recognizes and binds ABA by the common gate-latch-lock mechanism resembling the Arabidopsis ABA receptors, but the ABA binding pocket in FePYR1 shows discrepant residues resulting in different binding affinity to ABA. Structure-guided alterations of amino acid residues in FePYR1 generated ABA receptor variants with significantly increased ABA binding affinity. Expression of FePYR1 in Arabidopsis conferred enhanced drought resistance in the transgenic plants. These findings provided detailed information about FePYR1 and demonstrated that structure-assisted engineering could create superior ABA receptors for improving plant drought resistance. The detailed structural information of FePYR1 would also assist future rational design of small molecules targeting specific ABA receptors in economically important plant species.

Two Chloroplast Proteins Suppress Drought Resistance by Affecting ROS Production in Guard Cells.

Chloroplast as the site for photosynthesis is an essential organelle in plants, but little is known about its role in stomatal regulation and drought resistance. In this study, we show that two chloroplastic proteins essential for thylakoid formation negatively regulate drought resistance in Arabidopsis (Arabidopsis thaliana). By screening a mutant pool with T-DNA insertions in nuclear genes encoding chloroplastic proteins, we identified an HCF106 knockdown mutant exhibiting increased resistance to drought stress. The hcf106 mutant displayed elevated levels of reactive oxygen species (ROS) in guard cells, improved stomatal closure, and reduced water loss under drought conditions. The HCF106 protein was found to physically interact with THF1, a previously identified chloroplastic protein crucial for thylakoid formation. The thf1 mutant phenotypically resembled the hcf106 mutant and displayed more ROS accumulation in guard cells, increased stomatal closure, reduced water loss, and drought resistant phenotypes compared to the wild type. The hcf106thf1 double mutant behaved similarly as the thf1 single mutant. These results suggest that HCF106 and THF1 form a complex to modulate chloroplast function and that the complex is important for ROS production in guard cells and stomatal control in response to environmental stresses. Our results also suggest that modulating chloroplastic proteins could be a way for improving drought resistance in crops.