The LbCas12ai-mTREX2 system enhances large deletions, and the LbCas12ai-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccharum spp. hybrids) is a major feedstock for biofuel production and the primary source of global table sugar; however, its highly polyploid and complex genome constrains the effective deployment of functional genomics and precision breeding strategies. While CRISPR/Cas9-mediated genome editing has been reported in sugarcane, Cas12a-based editing remains unexplored despite its advantages in targeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbCas12ai-R) and LbCas12a-RRV (LbCas12ai-RRV) variants and assessed the impact of Mus musculus Three prime repair exonuclease 2 (mTREX2) co-expression on editing efficiency and deletion profiles. Editing efficiencies ranged from 31 to 85%, where LbCas12ai-RRV consistently outperformed LbCas12ai-R in both overall editing efficiency and allelic editing efficiency. Co-expression of the mTREX2 shifted the mutation spectrum toward larger deletions, increasing the frequency of moderate-to-large deletions. The ≥ 51 bp deletion class increased ~7-fold in both variants. Capillary electrophoresis-based genotyping provided a cost-effective, high-throughput alternative to next-generation sequencing (NGS) and accurately predicted allelic editing efficiency and indel size. This first report of CRISPR/Cas12a-mediated genome editing in sugarcane provides a framework for future engineering of AT-rich cis-regulatory elements in the polyploid sugarcane genome.

Alternative splicing (AS) is a pivotal post-transcriptional mechanism for plant thermal adaptation, yet its dynamics in heat-tolerant woody species remain poorly understood. Here, we map the temperature-resolved AS landscape of the highly thermotolerant woody plant Rhododendron hainanense by combining Iso-Seq with RNA-seq. Transcriptomic profiling revealed a graded regulatory architecture: moderate heat (37 °C) elicited a targeted proteostatic response, whereas severe heat (42 °C) provoked a massive transcriptome expansion. Notably, this global AS surge coincided with a widespread downregulation of core splicing factors, a correlation suggesting that a more permissive splicing environment may contribute to the rapid generation of diverse stress-responsive isoforms. The heat shock protein (HSP) family emerged as the principal hub of this AS-associated diversification; the core chaperone genes ClpB1, HSP83, and HSP70 generated the greatest transcript complexity, with HSP83 alone producing 29 distinct AS transcripts that resolved into three temperature-dependent expression groups. Within the heat shock factor network, HSFA2 showed the strongest and most sustained induction, and DAP-seq identified in vitro HSFA2 binding at the promoters of ClpB1, HSP83, and HSP70, supporting these core chaperones as putative direct DNA-binding targets. Functional complementation in Arabidopsis further showed that three structurally distinct HSFA2 splice variants each restored thermotolerance to the hsfa2 mutant and consistently induced expression of the core ATP-dependent chaperone arm, suggesting that this central chaperone-induction capacity is retained across the tested splice variants in this heterologous system. Together, these findings provide a comprehensive atlas of temperature-modulated AS in a thermotolerant woody plant and support a model in which splicing-mediated diversification of the HSP chaperone network contributes to heat adaptation, offering post-transcriptional insights for breeding climate-resilient crops.

The oil extracted from the mesocarp of oil palm (Elaeis guineensis, Jacq.) drupes is the world's leading source of vegetable oil. Oil palm cultivation requires large amounts of mineral nutrients supplied through fertilization, representing a major production cost. Among essential nutrients, potassium (K[+]) plays a critical role in plant growth and fruit development. However, the mechanisms underlying K[+] transport and its relationship with yield remain poorly understood. Here, we identified and characterized EgKT2-1, a Shaker-like potassium channel belonging to the AKT2-like subfamily. Quantitative PCR analyses showed that EgKT2-1 is expressed in leaves, meristem, and during early fruit development, with higher transcript abundance during early fruit development coinciding with increased K[+] accumulation. In situ hybridization localized EgKT2-1 transcripts to the root cortex and phloem tissues, leaf palisade parenchyma, and mesocarp cells during early fruit development. These expression patterns are consistent with a potential contribution of EgKT2-1 to K[+] transport and allocation between source and sink tissues. Although sequence similarity places EgKT2-1 within the weakly rectifying AKT2-like subfamily, electrophysiological characterization in Xenopus laevis oocytes revealed inwardly rectifying channel activity, suggesting a potential role in K[+] uptake. EgKT2-1 expression was higher in an oil palm progeny C3 which exhibited greater K[+] allocation to aerial organs and greater oil yield. Together, these findings identify EgKT2-1 as an inwardly rectifying K[+] channel potentially involved in K[+] transport and redistribution during oil palm development and suggest a correlation between EgKT2-1 expression, K[+] allocation, and oil yield variation.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis, but its role in grapevine cold tolerance is not well understood. Here we identified 23 VvCAD members from the 'Pinot Noir' genome and focused on VvCAD15-like, a Group II member whose expression was induced by cold. Transient overexpression of this gene in grape calli and plantlets increased lignin content, antioxidant enzyme activities (CAT, POD, SOD), and proline level, while decreased MDA, H2O2, and membrane permeability. It also upregulated VvCBF1/3 and VvICE1a. Transcriptome data implied potential links between VvCAD15-like and phenylpropanoid, flavonoid, and hormone signaling pathways. Yeast one-hybrid and dual-luciferase reporter assays identified three transcription factors-VvbHLH112, VvbHLH95, and VvNF-YA7-that could bind and activate the VvCAD15-like promoter under cold treatment. Among them, VvbHLH112 had transcriptional activation activity, and VvbHLH95 was closely related to AtICE1. Together, these data indicate that VvCAD15-like promotes cold tolerance possibly through cell wall reinforcement, ROS scavenging, and CBF pathway activation. This is the first report on VvCAD15-like function in grape cold tolerance, revealing its non-canonical role beyond lignin biosynthesis. It should be noted, however, that these results came from transient expression systems, and stable transgenic lines are needed to confirm its function in whole plants. Our work provides useful gene resources for cold tolerance breeding in grapevines.