Yongguo Zhao

Anthracnose disease, caused by Colletotrichum species, poses a significant threat to global litchi production, yet the molecular mechanisms governing host resistance remain poorly understood. To dissect the genetic basis of anthracnose resistance, we employed a comparative transcriptomic approach using two contrasting cultivars: ‘YuJinQiu’ (DR, disease-resistant genotype) and ‘BaiTangYing’ (DS, disease-susceptible genotype). Field evaluations and controlled infection assays demonstrated evident phenotypic divergence, with DR exhibiting delayed disease progression and 43.7% smaller lesion areas compared to DS at 72 h post-inoculation (hpi). Time-resolved RNA sequencing (0–72 hpi) revealed genotype-specific transcriptional dynamics, where DR displayed fewer differentially expressed genes (DEGs; 819–1457) compared to DS (5195–5735), suggesting a more targeted and efficient defense response. Functional enrichment analyses highlighted rapid activation of innate immunity pathways in DR, including pattern-triggered immunity, MAPK signaling, and jasmonic acid/ethylene biosynthesis, whereas DS prioritized cell wall modification and compensatory secondary metabolic processes. Weighted gene co-expression network analysis (WGCNA) pinpointed three modules tightly linked to anthracnose resistance, enriched for receptor-like kinases (RLKs), nucleotide-binding leucine-rich repeat (NLR) proteins, and phenylpropanoid biosynthesis genes. Hub regulators, including WRKY transcription factors (e.g., WRKY33), ubiquitin ligases, and pathogenesis-related proteins (PR1, PR5), were identified as central coordinators of defense signaling. Strikingly, DR exhibited sustained upregulation of effector-triggered immunity markers, particularly nucleotide-binding leucine-rich repeat (NLR) genes, and early accumulation of phytoalexins, correlating with pathogen suppression. Experimental validation via qRT-PCR confirmed the reliability of transcriptomic data. Our study unravels the multilayer regulatory network underlying litchi anthracnose resistance, providing not only a mechanistic model of cultivar-specific responses but also a robust gene toolkit for accelerating the development of resistant cultivars through marker-assisted breeding.

WRKY transcription factors serve as key regulators in plants, playing important roles in growth and development, secondary metabolism, and stress responses. Here, a comprehensive genome-wide analysis identified 58 WRKY genes (LcWRKYs) in litchi for the first time. All LcWRKY proteins were predicted to be hydrophilic and localized in the nucleus. Phylogenetic analysis classified them into three major groups (Groups I, II, and III), with a pronounced expansion of Group II, which contained 42 members divided into five subgroups. Members within the same phylogenetic clade exhibited highly similar exon–intron structures and conserved motif compositions, indicating strong evolutionary conservation. LcWRKYs were unevenly distributed across the litchi chromosomes, with chromosome 3 showing the highest gene density. Collinearity analysis suggested that both segmental and tandem duplications contributed to the evolutionary expansion of this family. Notably, promoter cis-acting element analysis revealed that LcWRKYs are enriched with light-responsive, hormone-responsive (e.g., ABA, MeJA, SA), and stress-responsive elements, suggesting their potential involvement in integrating light signaling, hormonal pathways, and environmental stress responses. Integrative expression analysis further revealed that multiple LcWRKYs were significantly up-regulated during the middle and late stages of fruit development in cultivars such as ‘Feizixiao’ and ‘Nuomici’. Consistent with these patterns, qRT-PCR validation demonstrated a pronounced induction of four representative genes (LITCHI004628.m1, LITCHI018082.m1, LITCHI021964.m1, and LITCHI030932.m1) at 40 days post-anthesis, followed by gene-specific expression trajectories at later stages, indicating their potential involvement in regulating fruit development, particularly during the mid-developmental stage. Altogether, the results of this study provide insight into the expansion and potential functional diversification of WRKY transcription factors in litchi and identify candidate regulators associated with fruit development, offering valuable targets for future functional studies and genetic improvement.

Caleosins (CLOs) or peroxygenases (PXGs), a class of structural proteins of lipid droplets (LDs), comprise a small family of multifunctional proteins widely involved in oil accumulation, organ development, and stress responses. Despite the proposal of two clades termed H and L in Arabidopsis thaliana, their evolution in the order Brassicales has not been well established. In this study, the first genome-wide analysis of the caleosin family was conducted in papaya (Carica papaya), a Caricaceae plant without any recent whole-genome duplication (WGD). A high number of five members representing both H and L clades were identified from the papaya genome. Further identification and comparison of 68 caleosin genes from 14 representative plant species revealed seven orthogroups, i.e., H1–4 and L1–3, where H1 and L1 have already appeared in the basal angiosperm Amborella trichopoda, supporting their early divergence before angiosperm radiation. Five CpCLO genes belong to H1 (1) and L1 (4), and extensive expansion of the L1 group was shown to be contributed to by species-specific tandem and transposed duplications, which may contribute to environmental adaptation. Orthologous and syntenic analyses uncovered that lineage-specific expansion of the caleosin family in Brassicales relative to A. trichopoda was largely contributed to by tandem duplication and recent WGDs, as well as the ancient γ whole-genome triplication (WGT) shared by all core eudicots. Independent gain or loss of certain introns and apparent expression divergence of caleosin genes were also observed. Tissue-specific expression analysis showed that CpCLO2 and −5 are constitutively expressed, whereas others appear to be tissue-specific, implying function divergence. Interestingly, the H-forms CpCLO1 and RcCLO1 were shown to exhibit similar expression profiles to most oleosin genes that are preferentially expressed oil-rich tissues such as seeds/endosperms, shoots, and calluses, implying their putative involvement in oil accumulation, as observed in Arabidopsis. The results obtained from this study provide a global view of CpCLO genes and insights into lineage-specific family evolution in Brassicales, which facilitates further functional studies in papaya and other non-model species.

Transposable elements (TEs) are abundant components of plant genomes, yet their transcriptional activity and potential biological roles remain underexplored, especially under environmental stress conditions. This study investigates the transcriptional dynamics of TEs in Brassica napus during drought stress in seed development, aiming to uncover their contributions to stress responses and seed germination. RNA-seq data were analyzed for TE transcriptional activity in wild-type (WT) and BnaABI5 CRISPR-edited mutant lines of B. napus. A comprehensive computational pipeline was used to identify and characterize TE-derived transcripts, including protein-coding and long non-coding RNAs (lncRNAs). Functional annotation was performed for protein-coding TE transcripts located in intergenic regions to predict their involvement in biological processes. Out of 212,800 TEs identified in the B. napus genome, 17,547 were transcriptionally active, yielding 15,808 protein-coding transcripts and 1,739 lncRNAs. Among these, 65 protein-coding TE transcripts were identified as transposase genes, while 860 transcripts were predicted to represent novel genes derived from transposon regions, potentially participating in monocarboxylic acid metabolic processes. Specific to drought stress responses during seed germination, 128 protein-coding TE transcripts (including 5 transposases) and 37 lncRNAs were differentially expressed. Notably, the lncRNA transcripts MSTRG.108925.4 and MSTRG.109003.7 were implicated in regulating the PHD finger protein ALFIN-LIKE 1 (BnA10g0418090), contributing to drought tolerance mechanisms. This study highlights the functional relevance of TE transcription in the context of drought stress during seed germination, providing novel insights into TE-derived genes and lncRNAs as potential regulators of stress responses. These findings expand the understanding of TE biology in plants and offer valuable resources for future efforts to identify drought-resistant genes in B. napus.

Key message A WRI-like gene (CeWRI3) homologous to AtWRI3/4 was isolated from oil-rich Cyperus esculentus tubers, which was shown to function in TAG accumulation through activating CeOLE2. Abstract In oilseeds, WRI1 has proven to be a master regulator in the transcriptional control of genes involved in fatty acid biosynthesis. By contrast, no evidence is available for WRI1 and its homologs in regulating genes associated with triacylglycerol (TAG) biosynthesis. In this study, we present the characterization of a WRINKLED (WRI)-like gene from tigernut (Cyperus esculentus L., Cyperaceae), a rare example accumulating high levels of TAGs in underground tubers. This gene was named CeWRI3 for its homology to AtWRI3/-4 in Arabidopsis thaliana. Subcellular localization analysis in Nicotiana benthamiana and transactivation assay in yeast revealed that CeWRI3 indeed functions as a transcription factor with nuclear localization and transcriptional activation activity. Further qRT-PCR analysis showed that CeWRI3 exhibits a constitutive expression pattern and its transcripts are positively correlated with TAG accumulation during tuber development. Moreover, transient overexpression of CeWRI3 in N. benthamiana leaves could significantly enhance the TAG content, implying its contribution to TAG accumulation in tigernut tubers. Correspondingly, yeast one-hybrid and dual-luciferase reporter assays revealed that CeWRI3 could activate CeOLE2, but not CeOLE5 and CeDGAT2b, another two key genes related to TAG biosynthesis. Our findings emphasize the potential application of CeWRI3 in improving oil production of vegetative tissues.

Tigernut (Cyperus esculentus L.), an oil-rich tuber plant of the Cyperaceae family, is typical for the naturally high content of oleic acid. However, to date, genes contributing to oil composition have not been well characterized. In this study, the first genome-wide analysis of tigernut genes encoding membrane-bound fatty acid desaturases (FADs), the key contributors to oil composition, is presented. According to phylogenetic analysis, ten members identified from the tigernut genome were assigned into seven out of eight evolutionary groups as defined in Arabidopsis thaliana, i.e., FAD2 (3), FAD6 (1), FAD3 (1), FAD7 (1), FAD4 (1), DES (1), and SLD (2). In contrast to the absence of an FAD5 homolog, FAD2 and SLD in tigernut were shown to have expanded via tandem and dispersed duplications, respectively. Comparison of 285 members from 29 representative plant species resulted in 11 orthogroups, where FAD2a, FAD6, FAD7, FAD3, FAD4, FAD5, DES, and SLD1 were shown to have already appeared in the ancestor of seed plants. Significantly, orthologous and syntenic analyses revealed that loss of FAD5 and expansion of SLD in tigernut are lineage-specific, occurred sometime before the radiation of core monocots, in contrast to species-specific expansion of FAD2. Moreover, though no syntenic relationship was observed between CeFAD genes, our comparative genomics analyses indicated that FAD3 and -7 are more likely to arise from segmental duplication. Structural variation and expression divergence of CeFAD genes were also observed. Gain of introns in CeFAD4, CeSLD1, and CeSLD2 was shown to be lineage-specific, occurred sometime before Cyperaceae-Juncaceae split. Tissue-specific expression analysis revealed that CeFAD2-1, CeFAD6, and CeFAD7 were constitutively expressed, whereas others were tissue-specific. Among five paralogs identified, CeFAD2-1 and CeSLD1 have evolved to be two dominant members. Putative roles of CeFAD2-1 in oil accumulation are supported by 1) exhibited an expression pattern positively associated with oil accumulation during tuber development; 2) were expressed more in tubers than their orthologs in C. rotundus. Additionally, in contrast to high expression of CrFAD3, transcript levels of CeFAD3 in tubers were fairly low, which may explain the distinct α-linolenic acid content between these two close species. Our findings provide a global view of CeFAD genes, which not only highlights lineage-specific evolution of the family, but also provides valuable information for further functional analysis and genetic improvement in tigernut.

LEA_5 domain-containing proteins constitute a small family of late embryogenesis-abundant proteins that are essential for seed desiccation tolerance and dormancy. However, their roles in non-seed storage organs such as underground tubers are largely unknown. This study presents the first genome-scale analysis of the LEA_5 family in tigernut (Cyperus esculentus L.), a Cyperaceae plant producing desiccation-tolerant tubers. Four LEA_5 genes identified from the tigernut genome are twice of two present in model plants Arabidopsis thaliana and Oryza sativa. A comparison of 86 members from 34 representative plant species revealed the monogenic origin and lineage-specific family evolution in Poales, which includes the Cyperaceae family. CeLEA5 genes belong to four out of five orthogroups identified in this study, i.e., LEA5a, LEA5b, LEA5c, and LEA5d. Whereas LEA5e is specific to eudicots, LEA5b and LEA5d appear to be Poales-specific and LEA5c is confined to families Cyperaceae and Juncaceae. Though no syntenic relationship was observed between CeLEA5 genes, comparative genomics analyses indicated that LEA5b and LEA5c are more likely to arise from LEA5a via whole-genome duplication. Additionally, local duplication, especially tandem duplication, also played a role in the family expansion in Juncus effuses, Joinvillea ascendens, and most Poaceae plants examined in this study. Structural variation (e.g., fragment insertion) and expression divergence of LEA_5 genes were also observed. Whereas LEA_5 genes in A. thaliana, O. sativa, and Zea mays were shown to be preferentially expressed in seeds/embryos, CeLEA5 genes have evolved to be predominantly expressed in tubers, exhibiting seed desiccation-like accumulation during tuber maturation. Moreover, CeLEA5 orthologs in C. rotundus showed weak expression in various stages of tuber development, which may explain the difference in tuber desiccation tolerance between these two close species. These findings highlight the lineage-specific evolution of the LEA_5 family, which facilitates further functional analysis and genetic improvement in tigernut and other species.

Plant oils rich in oleic acid (OA) are attracting considerable attention for their high nutritional value and significant industrial potential. Stearoyl-acyl carrier protein desaturases (SADs) are a class of soluble desaturases that play a key role in OA accumulation in plants. In this study, the first genome-wide characterization of the SAD gene family was conducted in tigernut (Cyperus esculentus L. var. sativus Baeck., Cyperaceae), an oil-rich tuber plant typical for its high OA content. Six SAD genes identified from the tigernut genome are comparative to seven reported in two model plants Arabidopsis thaliana and Oryza sativa, but relatively more than four were found in most Cyperaceae species examined in this study. A comparison of 161 SAD genes from 29 representative plant species reveals the monogenic origin and lineage-specific family evolution in Poales. C. esculentus SAD genes (CeSADs) were shown to constitute two evolutionary groups (i.e., FAB2 and AAD) and four out of 12 orthogroups identified in this study, i.e., FAB2a, FAB2b, FAB2c, and AAD1. Whereas FAB2a and AAD1 are widely distributed, FAB2b and FAB2c are specific to Cyperaceae, which may arise from FAB2a via tandem and dispersed duplications, respectively. Though FAB2d and AAD2 are also broadly present in monocots, they are more likely to be lost in the Cyperaceae ancestor sometime after the split with its close family, Juncaceae. In tigernut, FAB2a appears to have undergone species-specific expansion via tandem duplication. Frequent structural variation and apparent expression divergence were also observed. Though FAB2a and AAD1 usually feature two and one intron, respectively, gain of certain introns was observed in CeSAD genes, all of which have three introns. Despite recent expansion of the FAB2 group, CeFAB2-1 has evolved into the dominant member that was highly and constitutively expressed in all tested organs. Moreover, CeFAB2-1, CeAAD1, as well as CeFAB2-5 have evolved to be predominantly expressed in tubers and thus contribute to high OA accumulation. These findings highlight lineage-specific evolution of the SAD family and putative roles of CeSAD genes in tuber oil accumulation, which facilitate further functional analysis and genetic improvement in tigernut and other species.

Drought constitutes a noteworthy abiotic stressor, detrimentally impacting seed germination, plant development, and agricultural yield. In response to the threats imposed by climate change and water paucity, this study examined the morphological divergence and genetic governance of drought resilience traits at the germination stage in 196 rapeseed (Brassica napus L.) lines under both normal (0 MPa) and drought-induced stress (−0.8 MPa) scenarios. Our study showed that the composite drought tolerance D value is a reliable index for identifying drought resilience. Through a genome-wide association study (GWAS), we uncovered 37 significant SNP loci and 136 putative genes linked to drought tolerance based on the D value. A key discovery included the gene BnaA01g29390D (BnNCED3), encoding 9-cis-epoxycarotenoid dioxygenase, which exhibited significantly heightened expression levels in drought-resistant accessions (p < 0.01), underscoring its potential as a positive drought stress regulator and a suitable candidate for genetically enhancing drought resilience. Moreover, we pinpointed four stress-reactive transcription factors (BnaA07g26740D, BnaA07g26870D, BnaA07g26910D, and BnaA07g26980D), two E3 ubiquitin-protein ligases (BnaA05g22900D and BnaC06g28950D), two enzymes (BnaA01g29390D and BnaA03g48550D), and two photosystem-associated proteins (BnaA05g22950D and BnaC06g28840D) as vital components in drought response mechanisms. The construction of a regulatory network reveals an ABA-dependent pathway (NCED3/RGLG5/IDD14) that contributes to drought tolerance in rapeseed seedlings, alongside the involvement of a drought avoidance strategy (APRR6/PHYB). The SNPs and genes unveiled in this study offer a substantial theoretical foundation for subsequent investigations targeting genetic improvement for drought resilience during seed germination in rapeseed.

Main conclusion CeOLE6, a tuber-specific gene in tigernut, encodes a diverged SH oleosin that functions in oil accumulation via homo and heteromultimerization. Abstract Tigernut (Cyperus esculentus L.) is a rare example accumulating high levels of triacylglycerols (TAGs) in underground tubers; however, the mechanism underlying is poorly understood. Given essential roles of oleosins (OLEs) in oil accumulation, in this study, structural and functional analyses were conducted for CeOLE6, an oleosin gene preferentially expressed in tigernut tubers. Phylogenetic analysis revealed that CeOLE6 encodes a diverged oleosin in Clade SH, which also includes CeOLE4 and -5. Further synteny analysis and sequence comparison indicated that CeOLE6 is more likely to be a whole-genome duplication (WGD) repeat of CeOLE4, which underwent rapid evolution and deletion of the typical C-terminal insertion for SHs. Nevertheless, CeOLE6 retains the capacity of oligomerization and oil accumulation, because (i) CeOLE6 could not only interact with itself but also with CeOLE2 and -5, two tuber-dominant members belonging to Clades SL and SH, respectively, and (ii) overexpressing CeOLE6 in tobacco leaves could significantly enhance the TAG content. Though CeWRI1 exhibits a similar expression pattern as CeOLE6 during tuber development, both CeWRI1 and -3 could not activate the CeOLE6 promoter, implying that they are not transcription factors contributing tuber-specific activation of CeOLE6. These findings not only provide insights into CeOLE genes in tuber oil accumulation, but also lay a foundation for further genetic improvement in tigernut and other species.