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CRISPR/Cas is a simple, robust, versatile tool for plant biology studies and precision plant breeding. However, establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa (Medicago sativa L.), the most important forage legume worldwide, remains a formidable challenge. Here, we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves. We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system. Subsequently, we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA (PTG) expression cassettes, each consisting of three tRNA-sgRNA units, to simultaneously edit three or four alfalfa genes, coupled with the visual reporter RH1 or RUBY. This toolkit showed efficient multiplex editing in the hairy root system with visual selection. We successfully obtained regenerated, red-colored shoots resulting from the stable transformation of alfalfa. These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa. Our improved CRISPR/Cas9 multiplex system enables convenient, high-efficiency multiplex genome editing in alfalfa, providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.

RXLR effectors are pathogenic factors secreted from oomycetes to manipulate the immunity of the host. Typical RXLR effectors contain an RXLR-dEER motif at the N-terminus, whereas atypical RXLRs show variations on this motif. The oomycete Phytophthora cactorum is known to infect over 200 plant species, resulting in significant agricultural economic losses. Although genome-wide identification and functional analyses of typical RXLRs from P. cactorum have been performed, little is known of atypical PcaRXLRs. Here, we identified RXLRs, both typical and atypical, in P. cactorum and compared them with those of other oomycete pathogens. Fewer RXLRs were identified in P. cactorum compared with other Phytophthora species, possibly due to fewer duplication events of RXLRs. In contrast, the percentage of atypical RXLRs was higher in P. cactorum than in other species, suggesting significant roles in P. cactorum pathogenesis. Analysis of RXLR gene expression showed that most were transcribed, suggesting their functionality. Transient expression of two atypical RXLRs in Nicotiana benthamiana showed that they induced necrosis dependent on host SGT1 and HSP90. Furthermore, two additional atypical RXLRs suppressed the defense response in N. benthamiana and promoted P. cactorum infection. These results demonstrate the vital role of atypical RXLRs in P. cactorum and provide valuable information on their evolutionary patterns and interactions with host plants.

The heat stress (HS) response in plants involves complex processes at the molecular, cellular, and whole-organism levels. Sensitivity to HS differs based on the species and developmental stage of the plant, making it challenging to define HS and its impacts. Efforts to enhance HS tolerance by traditional breeding are constrained by limited genetic resources, but understanding the mechanisms that regulate HS responses can enable efforts to improve heat tolerance by precision breeding and gene editing. Here, we review recent research on the effects of HS on major cereal crops at different developmental stages and identify key genes potentially involved in the HS response, offering insight for precision molecular breeding. Additionally, we discuss the use of favorable natural variants and gene editing to improve crop tolerance to HS, emphasizing the value of alleles involved in thermomemory, combined stress tolerance, and the stress response–growth balance. This review aims to summarize recent advancements in understanding HS responses in crops, highlighting potential avenues for generating heat-tolerant crops.

Plant metabolites are crucial for the growth, development, environmental adaptation, and nutritional quality of plants. Plant metabolomics, a key branch of systems biology, involves the comprehensive analysis and interpretation of the composition, variation, and functions of these metabolites. Advances in technology have transformed plant metabolomics into a sophisticated process involving sample collection, metabolite extraction, high-throughput analysis, data processing, and multidimensional statistical analysis. In today’s era of big data, the field is witnessing an explosion in data acquisition, offering insight into the complexity and dynamics of plant metabolism. Moreover, multiple omics strategies can be integrated to reveal interactions and regulatory networks across different molecular levels, deepening our understanding of plant biological processes. In this review, we highlight recent advances and challenges in plant metabolomics, emphasizing the roles for this technique in improving crop varieties, enhancing nutritional value, and increasing stress resistance. We also explore the scientific foundations of plant metabolomics and its applications in medicine, and ecological conservation.

The embryo spot trait leads to a deep purple or reddish coloration at the base of the cotyledons of the embryo, visible on both sides of flat potato (Solanum tuberosum) seeds. This trait has long been used by potato researchers and breeders as a morphological marker during dihaploid induction. The formation of embryo spots reflects the accumulation of anthocyanins, but the genetic basis of this trait remains unclear. In this study, we mapped the embryo spot trait to a 6.78-Mb region at the end of chromosome 10 using an F₂ population derived from a cross between spotted and spotless plants. The recombination rate in the candidate region is severely suppressed, posing challenges for the map-based cloning of the underlying gene and suggesting large-scale rearrangements in this region. A de novo genome assembly of the spotted individual and a comparative genomic analysis to the reference genome of spotless potato revealed a 6.49-Mb inversion present in the spotted plant genome. The left breakpoint of this inversion occurred in the promoter region of an R2R3 MYB transcription factor gene that is highly expressed in the cotyledon base of spotted embryos but is not expressed in that of spotless embryos. This study elucidated the genetic basis for embryo spot formation in potato and provides a foundation for future cloning of the causative gene.

Legumes, such as peas, beans, and alfalfa, have evolved a remarkable ability to establish root nodule symbioses with nitrogen-fixing soil bacteria to fulfill their nitrogen needs. This partnership is characterized by a high degree of specificity, occurring both within and between host and bacterial species. Consequently, nodulation capacity and nitrogen-fixing efficiency vary significantly among different plant–bacteria pairs. The genetic and molecular mechanisms regulating symbiotic specificity are diverse, involving a wide array of host and bacterial genes and signals with various modes of action. Understanding the genetic basis of symbiotic specificity could enable the development of strategies to enhance nodulation capacity and nitrogen fixation efficiency. This knowledge will also help overcome the host range barrier, which is a critical step toward extending root nodule symbiosis to non-leguminous plants. In this review, we provide an update on our current understanding of the genetics and evolution of recognition specificity in root nodule symbioses, providing more comprehensive insights into the molecular signaling in plant–bacterial interactions.

The transition from vegetative to reproductive growth is a vital step for the reproductive success of plants. In Arabidopsis thaliana, LEAFY (LFY) plays crucial roles in inflorescence primordium and floral organ development, but little is known about the roles of its homologs in crop plants such as soybean (Glycine max). Here, we investigated the expression patterns and functions of the two LFY genes (LFY1 and LFY2) in soybean. Both genes were predominantly expressed in unopened flowers and the shoot apical meristem, with LFY2 having the higher transcript abundance. In an in situ hybridization assay, LFY genes produced strong signals in the floral meristem. We next generated lfy1 and lfy2 knockout lines. The lfy2 mutants showed obvious changes in floral organ morphology, but the lfy1 mutants showed no obvious changes in floral organ morphology or pod development. The lfy1 lfy2 double mutants displayed more serious defects in floral organ development than lfy2, resulting in complete sterility. Gene expression analysis revealed differences in expression of the A-class APETALA (AP) genes AP1a and AP1b in the double mutant lines. These results suggest that LFY2 plays an important role in floral organ formation in soybean by regulating the expression of homeotic genes. Our findings increase the understanding of floral development, which could be useful for flower designs during hybrid soybean breeding.

The advent of genome editing technologies, particularly CRISPR/Cas9, has significantly advanced the generation of legume mutants for reverse genetic studies and understanding the mechanics of the rhizobial symbiosis. The legume–rhizobia symbiosis is crucial for sustainable agriculture, enhancing nitrogen fixation and improving soil fertility. Numerous genes with a symbiosis-specific expression have been identified, sometimes exclusively expressed in cells forming infection threads or in nitrogen-fixing nodule cells. Typically, mutations in these genes do not affect plant growth. However, in some instances, germline homozygous mutations can be lethal or result in complex pleiotropic phenotypes that are challenging to interpret. To address this issue, a rhizobia-inducible and cell-type-specific CRISPR/Cas9 strategy was developed to knock-out genes in specific legume transgenic root tissues. In this review, we discuss recent advancements in legume genome editing, highlighting the cell-type-specific CRISPR system and its crucial applications in symbiotic nitrogen fixation and beyond.

The plant hormone ethylene regulates plant growth, development, and stress responses. Recent studies on early signaling events following ethylene perception in rice (Oryza sativa) have identified MAO HU ZI 3 (MHZ3) as a stabilizer of the ethylene receptors ETHYLENE RESPONSE SENSOR 2 (OsERS2) and ETHYLENE RECEPTOR 2 (OsETR2). MHZ3 ensures the interaction of these receptors with CONSTITUTIVE TRIPLE RESPONSE 2 (OsCTR2), thereby maintaining OsCTR2 activity. Ethylene treatment disrupts the interactions within the MHZ3/receptors/OsCTR2 protein complex, leading to decreased OsCTR2 phosphorylation and the initiation of downstream signaling. Recent studies have established MHZ3 as the primary regulator and switch for OsCTR2 phosphorylation. In this review, we explore the role of MHZ3 in regulating ethylene signaling and highlight its effects on plant growth, development, and stress responses at the plant holobiont level.