The Gene Editing of Eukaryotic Translation Initiation Factor Binding Protein 3 and Its Potential Role in Rapid Cold Hardening of Two Invasive Fruit Flies
This work represents the first application of the CRISPR-Cas9 system in B. correcta, and provides insights into the molecular mechanisms underlying rapid cold hardening adaptation in B. correcta and a potential molecular marker for monitoring cold tolerance in field populations which could guide the development of novel control strategies that target the RCH adaptation pathways.
Abstract
Bactrocera dorsalis (Hendel) and Bactrocera correcta (Bezzi) are two globally concerning quarantine pests within the genus Bactrocera (Tephritidae). Both species inflict severe damage on the agricultural industry and international export trade. Their distribution range has expanded due to global warming, posing an increasing threat to fruit production. Heat shock proteins (HSPs), functioning as molecular chaperones, are known to contribute to temperature adaptation in insects. However, the cold adaptation regulatory mechanisms in these two Bactrocera species remain unclear. In this study, eIF4EBP3−/− mutations were established by the CRISPR-Cas9 system in both species. The survival rate of the mutations was significantly reduced with cold treatments, and qRT-PCR analysis indicated that eIF4EBP3 regulates the expression of several downstream heat shock proteins, suggesting that it may be involved in rapid cold hardening (RCH) adaptability in both fruit fly species. In addition, this work represents the first application of the CRISPR-Cas9 system in B. correcta, which provides methods to further studies on this species. Our results provide insights into the molecular mechanisms underlying rapid cold hardening adaptation in B. dorsalis and B. correcta and a potential molecular marker for monitoring cold tolerance in field populations which could guide the development of novel control strategies that target the RCH adaptation pathways.
Steroidal glycoalkaloids (SGAs) are toxic defense substances present in certain species of the Solanaceae, including major crops such as eggplant, tomato, and potato. GLYCOALKALOID METABOLISM 9 (GAME9) was first identified in tomato and potato as an APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor regulating key genes in SGA biosynthesis. However, the spatial effects of endogenous GAME9 in potato remain largely unexplored, particularly in tubers, highlighting a knowledge gap in understanding activation and induction of SGA biosynthesis in this important staple food crop. Here, we generated StGAME9 knockout mutants in potato via DNA-free CRISPR/Cas9. Compared to the wild type, knockout mutants contained significantly reduced SGA levels in leaves, and were almost free of SGAs in tubers. Notably, SGA accumulation remained minimal in the mutant tubers even under two SGA-inducing conditions; wounding and light exposure, indicating a loss of inducible SGA biosynthesis. Integrated transcriptomic and metabolomic characterization of knockout mutants (Stgame9) revealed extensive reprogramming of gene expression and metabolism, affecting not only SGA and sterol pathways but also a broader range of metabolic processes, with stress-related metabolic responses being attenuated in Stgame9 tubers. Despite these changes, Stgame9 plants displayed a normal growth phenotype under both greenhouse and field conditions. Our findings substantiate a pivotal role of GAME9 in potato for the regulation of basal and induced SGA biosynthesis. The results further indicate that StGAME9 is involved in the regulation of a broader, complex, and interconnected network along biosynthetic pathways, where potato metabolism exhibits substantial robustness and compensatory capacity to buffer the loss of StGAME9.
Ying Liu, Irene Merino, Lizel Potgieter et al.· Plant physiology and biochem...· 0 citations
Calcineurin B-like protein-interacting protein kinases (CIPKs) act as core regulators in plant calcium (Ca2+) signaling pathways and mediate abiotic stress adaptation. Mung bean (Vigna radiata L.) is an economically important legume that is widely grown in arid and semi-arid regions; however, drought stress significantly reduces its yield and quality. Despite this, genome-wide identification and functional analysis of the CIPK gene family have not been reported in mung bean, limiting our understanding of drought-resistance mechanisms and stress-tolerant variety breeding. In this study, 23 VrCIPK genes were identified from the mung bean genome, which were unevenly distributed across 6 chromosomes, with the remaining genes located on scaffolds. Phylogenetic analysis classified the VrCIPK family into five groups (A–E), characterized by high structural conservation of the N-terminal kinase and the C-terminal NAF/FISL regulatory domains. Gene Ontology annotation further indicated their conserved involvement in protein phosphorylation and calcium signal transduction. Collinearity analysis revealed that CIPK genes in mung bean and other species have relatively conserved evolutionary patterns. Segmental duplication contributed to VrCIPK gene family expansion, and most duplicated gene pairs underwent purifying selection during evolution. Gene structure and motif analyses showed that VrCIPK genes within the same group shared conserved structural features. Cis-acting element profiling revealed abundant hormone- and stress-responsive elements in VrCIPK promoters, indicating diverse transcriptional regulatory potential. Transcriptomics analysis and quantitative real-time PCR demonstrated that VrCIPK5 was significantly induced under drought stress. The heterologous overexpression of VrCIPK5 in tobacco conferred enhanced drought tolerance by promoting proline accumulation, increasing antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities, accelerating stomatal closure, and upregulating downstream stress-responsive genes (NtSOD, NtCAT, NtP5CS1, NtLEA5, and NtRD29A), thus alleviating reactive oxygen species overaccumulation and membrane lipid peroxidation. Collectively, these findings fill the research gap in the genome-wide identification and functional analysis of the CIPK gene family in mung bean and provide key genetic resources and theoretical support for breeding drought-resistant mung bean varieties.
L. Yin, Jiaqi Liao, Jie Gao et al.· Frontiers in Plant Science· 0 citations
Small heat shock proteins (Hsp20s) function as essential molecular chaperones in plant stress responses, yet their genome-wide characterization in sunflower (Helianthus annuus L.) remains lacking and their functional role in heat response is also unknown. In this study, 65 HaHsp20 genes were identified in sunflower through a comprehensive genome-wide analysis based on the conserved ACD (α-crystallin) domain. The expansion of this family was primarily driven by whole-genome duplication (WGD) or segmental duplication events, with the CI subfamily (20 members) representing the most significantly expanded lineage-specific clade. While all HaHsp20 proteins harbor the conserved α-crystallin domain (ACD), they exhibit diverse molecular weights (11.31–53.35 kDa), isoelectric points (4.71–9.75), and subcellular localization patterns. Promoter cis-regulatory element analysis revealed a predominance of ABA and MeJA-responsive elements but only two canonical heat shock elements. Transcriptome and RT-qPCR analyses revealed that most HaHsp20 genes are responsive to heat stress, with seven HaHsp20 genes exhibiting extremely upregulated expression (more than 1000-fold) after 10 h of 45 °C treatment. Among these, HaHsp21.59 and HaHsp25.91 showed an increase of over 4000-fold in expression. These findings provide a comprehensive foundation for understanding the evolutionary history and expression dynamics of the HaHsp20 family in sunflower, and highlight HaHsp21.59 and HaHsp25.91 as promising candidate genes for future functional validation of their potential roles in heat stress tolerance.
Yushan Liu, Shurui Dong, Qian Zhang et al.· International Journal of Mol...· 0 citations
The RING-type E3 ubiquitin ligase plays a significant role in plant responses and adaptations to abiotic stresses such as drought. However, few studies have explored the role of E3 ubiquitin ligases in potato drought stress, especially DRIP1. In this study, 172 StHCa genes were identified across the potato genome. These genes were unevenly distributed on twelve chromosomes and divided into six subclades (group I–VI). The molecular weight of potato HCa proteins ranges from 5445.38 to 143,293.69 Da. More than half of them are acidic proteins and most are unstable. There are 161 hydrophilic proteins, and the subcellular localization analysis indicated that they were mainly located in the nucleus. The co-linearity analysis of StHCa genes showed that 172 genes underwent 40 tandem duplications and 28 segmental duplication events. Potato and tomato share a recent common ancestor and exhibit highly similar evolutionary trajectories. Promoter sequence analysis of the StHCa family identified abundant cis-acting elements associated with light signal transduction, hormone responses, plant growth and development, and abiotic stress responses. These results suggest that the StHCa genes may play important regulatory roles in different environmental signals and developmental stages. Expression profiling revealed that StDRIP1 exhibited higher transcript levels in potato roots than in stems and leaves, and its expression was significantly induced by drought stress. Physiological phenotyping demonstrated that StDRIP1-overexpressing (OE) plants displayed reduced root growth compared with wild-type (WT) plants, with decreases in root length, total root area, total root volume, and root vitality. Under 20% PEG-6000-simulated drought stress, the root expression level of StDRIP1 was higher in OE lines than in WT plants. Furthermore, StDRIP1 overexpression suppressed the activities of antioxidant enzymes (POD, CAT, and SOD) and weakened their osmotic adjustment ability by reducing proline (Pro) accumulation. At the late stage of stress treatment (9 h), the SOD, POD, and CAT activities of OE plants were 5.0%, 19.9%, and 25.1% lower than those of WT plants, respectively. These physiological alterations exacerbated oxidative damage, as evidenced by increased malondialdehyde (MDA) content and elevated electrolyte leakage in OE plants. In summary, this study comprehensively characterized the StHCa gene family in potato, providing a valuable theoretical basis for elucidating the functional mechanism of StDRIP1 in modulating drought stress responses. Collectively, StDRIP1 acts as a negative regulator of potato drought tolerance through two primary mechanisms: (1) repressing root growth and weakening root vitality, thereby reducing the water absorption capacity of roots; and (2) diminishing antioxidant enzyme activities and impairing osmotic homeostasis, which further exacerbates oxidative damage under drought stress.
Xiaoyuan Liu, Xingyu Zhou, Haoran Wen et al.· Current Issues in Molecular...· 0 citations
In seed plants, putrescine, spermidine, and spermine are ubiquitously present, whereas a structural isomer of spermine, thermospermine (TSpm), is synthesized mainly in the vascular tissue. Initially identified in the bacterium Thermus thermophilus, TSpm was later shown to be synthesized in Arabidopsis thaliana by ACAULIS5 (ACL5). ACL5 gene homologs may have been acquired early in plant evolution via endosymbiotic gene transfer from a cyanobacterial ancestor. Loss-of-function acl5 mutants exhibit a dwarf phenotype and excessive vascular xylem formation. Subsequent studies, including analysis of suppressor-of-acl5 (sac) mutants, revealed that TSpm exerts a critical role in the repression of vascular xylem proliferation by acting in upstream open-reading-frame (uORF)-dependent translational regulation of specific mRNAs. A recent study revealed functional TSpm binding to the peptidyl transferase center of 25 S rRNA promoted by methylation of residue U2952. Like other polyamines, TSpm has also been shown to participate in stress responses, enhancing tolerance to salt, drought, heat, and pathogen challenges in multiple species. Collectively, TSpm represents a unique polyamine with dual roles in xylem development and stress adaptation, whose evolutionary origin and molecular mechanisms provide insights into the specialization of polyamine biology. Further studies in nonvascular plants and algae are needed to elucidate the ancestral functions of TSpm.
Sugarcane (Saccharum spp.) is a major sugar crop, yet its growth and yield are frequently limited by soil salinization. Receptor-like cytoplasmic kinases (RLCKs) act as central signaling hubs in plants, regulating growth, development, immunity, and abiotic stress responses. However, the evolution of the RLCK gene family in sugarcane and its role in salt tolerance remain unclear. Using the high-quality genome of elite cultivar 'Zhongzhe No.1', we identified 262 ScRLCK genes, grouped into 15 subfamilies, all of which contain a typical protein kinase domain. Synteny analysis revealed that whole-genome and segmental duplications were the primary drivers of ScRLCK family expansion, accounting for 67.9% duplication events, while purifying selection predominated during their evolutionary trajectory. T Transcriptome profiling revealed widespread differential expression of ScRLCK genes under hormonal treatments and abiotic stresses; notably, ScRLCK53 was strongly upregulated by salt and other stresses. Functional characterization confirmed nuclear localization of the ScRLCK53. Transgenic Arabidopsis lines displayed markedly enhanced seed germination rates and increased seedling fresh weight under salt stress. Moreover, these overexpression lines exhibited heightened sensitivity to jasmonic acid (JA), accompanied by elevated endogenous JA levels, pronounced upregulation of key JA signaling markers, and enhanced expression of canonical salt-responsive genes. This study represents the first comprehensive characterization of the RLCK gene family in sugarcane, elucidating its evolutionary features, expression dynamics and functional roles, and provides compelling evidence that ScRLCK53 modulates salt tolerance through activation of the JA signaling pathway, providing a theoretical foundation and valuable genetic resources for the genetic enhancement of stress tolerance in sugarcane.
Shichao Wang, Pingping Lin, Deng Wu et al.· Plant physiology and biochem...· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026
