Plant Biotechnology Journal最新文献

Rice has developed an efficient system for manganese (Mn) uptake, mediated by two distinct transporters, OsNramp5 and OsMTP9. These transporters exhibit polar localization at the root exodermis and endodermis; however, the mechanisms underlying their polar localization and their role in Mn uptake remain unclear. Here, we identified key amino acid residues critical for the polar localization of OsNramp5 at the distal side. Through analysis of chimeric proteins between OsNramp5 and its non-polar homologues, we found that the C-terminal cytosolic region of OsNramp5 is essential for its polar localization. Site-directed mutagenesis further revealed that aspartate 500 and four valine residues at positions 494, 495, 498 and 506 are crucial for polarity. Substitution of these valine residues with isoleucine, leucine, phenylalanine, or threonine partially or fully maintained polar localization, whereas substitution with alanine, serine, or asparagine resulted in loss of polarity. These findings suggest that β-branching and high hydrophobicity of amino acid side chains are likely required for OsNramp5 polarity. Furthermore, we found that adaptor protein 2-dependent clathrin-mediated endocytosis is not involved in the polar localization of OsNramp5. Finally, we provided experimental evidence showing the significant role of OsNramp5 polarity in efficient Mn uptake in rice; plants expressing non-polarly localized OsNramp5 exhibited reduced Mn uptake compared to those with polarly localized OsNramp5. In addition, we found that cadmium accumulation in shoots could be reduced by manipulating OsNramp5 polarity in combination with its overexpression, without a growth penalty.

Haploid induction coupled with genome editing (HI-Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI-Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post-pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin-associated domain-2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post-pollination heat treatment alone increased HER to 19.1% (up to 12-fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6-fold at the Waxy1 (Wx1) locus and 4.5-fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR-Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI-Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite-line genome editing.

Soil salinity is a major abiotic stress that threatens global crop productivity. Calcium (Ca²⁺) is a pivotal second messenger in plant stress signalling, but how Ca²⁺ sensors such as calmodulin (CaM) transcriptionally control ion homeostasis and salt tolerance remains largely understood in crops. Here, we identified a novel calmodulin-binding protein HvCBP60b, which is a negative regulator of salt resilience in salt-tolerant barley. We showed that HvCBP60b interacts with Ca²⁺ sensor HvCaM1 via its C-terminal domain in a Ca²⁺-dependent manner. Loss of either HvCBP60b or HvCaM1 promoted the uptake of K⁺, increased the K⁺/Na⁺ ratio, and markedly enhanced both growth performance and grain yield under salinity stress. Furthermore, HvCBP60b binds to the promoter of R2R3-type MYB transcription factor HvMYB77 through its N-terminal domain. HvMYB77 then transcriptionally reduces the expression of HvHAK5, a high-affinity K⁺ transporter essential for maintaining K⁺ homeostasis under salinity. Mutations in HvMYB77 or HvHAK5 compromise salt tolerance, whereas the salt-sensitive phenotype of hvhak5 is rescued in hvcbp60b-hvhak5 double mutants. Therefore, we uncover a novel Ca²⁺-responsive regulatory module CaM1-CBP60b-MYB77-HAK5 essential for K⁺ homeostasis and salt tolerance in barley, highlighting new targets of Ca²⁺ signalling for improving salt resilience in cereal crops.

Advancements in plant genome sequencing and assembly have enabled the production of increasingly accurate and contiguous genome sequences. Here, we present the chromosome-level assembly of the durum wheat (Triticum turgidum L. ssp. durum, cv. Svevo) reference genome produced using accurate long-reads, optical mapping and Hi-C. The new assembly (Svevo Rel.2.0) comprises 263 hybrid scaffolds with an N50 value of 112.3 Mb, arranged into 14 contiguous pseudomolecules spanning 10.4 Gb. The Svevo Rel.2.0 genome assembly was annotated using extensive short- and long-read RNA sequencing data obtained from 60 tissue/treatment combinations. The resulting annotation comprises 68 154 high-confidence protein-coding genes, which have been integrated into a comprehensive transcriptome atlas accessible through an eFP browser. Annotation was manually curated for storage protein gene families and for Leucine-Rich Repeat-Containing Receptor genes yielding 3763 LRR-CR loci. The genome assembly's accuracy and completeness were demonstrated by the correct reconstruction of the physical map of Tg1-B (Tenacious glumes 1), a locus controlling the free threshing trait located on chromosome 2B that was not assembled in the previous genome release (Svevo Rel.1.0). A wealth of 6621 QTLs/MTAs from the literature were mapped onto Svevo Rel.2.0 to identify QTL hotspots and trait-specific candidate genes. The ancestry of the durum genome to representative wild emmer populations from North-Eastern and Southern-Levant Fertile Crescent assessed by tracing haplotype transmission patterns revealed a clear mosaic pattern. This new durum reference genome, enhanced with advanced annotation and an expression atlas linked to QTLome data, is the most comprehensive tool available for durum wheat genomics.

Drought stress severely restricts rice productivity, necessitating strategies to enhance stress resilience. Here, we identify translation termination as a tunable regulatory node for stress adaptation. Using 25 rounds of SELEX, we isolated RTAR, an RNA aptamer that binds the rice eukaryotic release factor 1 (OseRF1) with nanomolar affinity. Structural and functional analyses revealed that RTAR targets the OseRF1 N-domain through a conserved pocket centered on Arg46, inhibiting stop-codon recognition and promoting translational readthrough in rice cell extracts. Under drought stress, RTAR co-localized with OseRF1 in rice protoplasts and accelerated its depletion, thereby reprogramming termination fidelity. Ribosome profiling (Ribo-seq) identified candidate genes, including OsDR8, with increased ribosome occupancy downstream of annotated stop codons, indicating a transcriptome-wide effect on translation termination. RTAR expression significantly enhanced drought tolerance in rice seedlings, as evidenced by increased survival, chlorophyll retention and reduced membrane injury. These findings were further validated in stable transgenic lines subjected to soil-based drought stress, where RTAR-expressing plants exhibited enhanced whole-plant drought resilience. This phenotype was accompanied by upregulation of drought-responsive genes and was abolished in OseRF1-R46A mutants, demonstrating the functional importance of the Arg46-centered binding pocket. Furthermore, GFP-tagging assays revealed higher-molecular-weight OsDR8 isoforms consistent with C-terminal extension, providing direct evidence of translational readthrough in vivo. Together, these results establish RTAR as a precise and reversible modulator of translation termination and identify OseRF1 as a promising target for engineering drought adaptation in crops.

Seed size plays an important role in determining crop yield, yet the genetic mechanisms underlying the regulation of this agronomic trait remain incompletely understood in soybean. Here, we perform a genome-wide association study mapping with seed length variations and identify a major quantitative trait locus (QTL) on chromosome 20, named SL20 (Seed Length 20). GmSL20 encodes a TGA-type bZIP transcription factor that controls soybean seed size and weight in natural populations. Knockout of GmSL20 suppresses seed enlargement and lipid biosynthesis in soybean, whereas increased expression of GmSL20 improves seed size and oil content. Further functional investigations reveal that GmSL20 directly activates GmST05 and GmZF351, thus promoting carbon partitioning to seeds. A super allele that arose from domestication confers elevated capacity for DNA binding and transcriptional activation to GmSL20. It correlates with larger seed size in haplotype analysis and enhances seed size and weight in elite soybean variety; however, it has not been widely fixed and spread through modern breeding. The role of GmSL20 to promote yield and quality underscores its potential as a valuable resource for soybean breeding programs seeking to improve both traits simultaneously.

Drought constitutes a major abiotic stress limiting alfalfa productivity. To elucidate the genetic basis of drought tolerance, a genome-wide association study (GWAS) was conducted in alfalfa, which identified a CBF/DREB1 family transcription factor designated MsCBF8. Utilizing an optimized CRISPR-Cas9 system, mutants were successfully generated in the complex autotetraploid background. Combined with overexpression and Arabidopsis complementation assays, this confirmed that MsCBF8 functions as a positive regulator of drought tolerance, with its overexpression enhancing drought tolerance without compromising plant growth. Physiological analyses revealed that MsCBF8 improves drought tolerance by coordinating antioxidant defence (elevated SOD and CAT activities, decreased H₂O₂ and MDA contents), dynamically regulating stomatal movement (increased water-use efficiency), and maintaining photosynthetic performance. At the molecular level, MsCBF8 directly and specifically binds to the GCCGAC cis-element in the MsGolS2 promoter and activates its expression. Silencing MsGolS2 via virus-induced gene silencing (VIGS) reduced drought tolerance and aggravated oxidative damage, providing the first functional evidence for the essential role of this gene in the drought response of alfalfa. This study systematically elucidates a novel MsCBF8-MsGolS2 regulatory module, offering crucial insights into the drought adaptation mechanisms in alfalfa and providing valuable genetic resources and breeding targets for developing high-yield, water-efficient and drought-tolerant alfalfa cultivars.

The escalating frequency and intensity of recent droughts threaten global crop production and food security. Although drought priming is crucial for plant drought resilience, the mechanisms imparting drought stress memory remain poorly understood, especially in crops such as tomato. This study elucidates the mechanisms of drought stress memory in tomato by integrating phenotypic screening with transcriptomic and proteomic analyses. Initial screening of 30 genotypes identified varieties with contrasting basal drought tolerance as indicated by gas exchange and drought damage index. Subsequent priming experiments revealed that acquired drought tolerance is independent of basal resistance, with both sensitive and tolerant genotypes exhibiting enhanced adaptation. Priming improved photosynthetic capacity and water use efficiency via optimised stomatal regulation. Moreover, elevated reactive oxygen species (ROS), particularly H₂O₂, was a key signalling molecule that initiated the drought memory establishment. This acquired drought tolerance was stress-specific, enhancing tolerance of tomato plants to subsequent drought but not heat stress. Integrative multi-omics analysis identified 518 memory-associated transcripts and pinpointed key candidate genes, including HSP90 (heat shock protein 90), WRKY26 and WRKY31 (WRKY transcription factor 26/31), which were linked to central pathways such as MAPK (mitogen-activated protein kinase) signalling and phenylpropanoid biosynthesis. The VIGS (Virus-Induced Gene Silencing) assay further demonstrated that WRKY26 functions as a negative regulator of acquired drought tolerance. Our work established a mechanistic framework for drought stress memory, highlighting ROS signalling and memory-specific gene networks. These insights, along with the generated datasets, provide valuable resources for the strategic breeding of climate-resilient tomato varieties.