Background: Timing is everything when it comes to the fitness outcome of a plant’s ecological interactions, and accurate timing is particularly relevant for interactions with herbivores or mutualists that are based on ephemeral emissions of volatile organic compounds. Previous studies of the wild tobacco N. attenuata have found associations between the diurnal timing of volatile emissions, and daytime predation of herbivores by their natural enemies. Results: Here, we investigated the role of light in regulating two biosynthetic groups of volatiles, terpenoids and green leaf volatiles (GLVs), which dominate the herbivore-induced bouquet of N. attenuata. Light deprivation strongly suppressed terpenoid emissions while enhancing GLV emissions, albeit with a time lag. Silencing the expression of photoreceptor genes did not alter terpenoid emission rhythms, but silencing expression of the phytochrome gene, NaPhyB1, disordered the emission of the GLV (Z)-3-hexenyl acetate. External abscisic acid (ABA) treatments increased stomatal resistance, but did not truncate the emission of terpenoid volatiles (recovered in the headspace). However, ABA treatment enhanced GLV emissions and leaf internal pools (recovered from tissue), and reduced internal linalool pools. In contrast to the pattern of diurnal terpenoid emissions and nocturnal GLV emissions, transcripts of herbivore-induced plant volatile (HIPV) biosynthetic genes peaked during the day. The promotor regions of these genes were populated with various cis-acting regulatory elements involved in light-, stress-, phytohormone- and circadian regulation. Conclusions: This research provides insights into the complexity of the mechanisms involved in the regulation of HIPV bouquets, a mechanistic complexity which rivals the functional complexity of HIPVs, which includes repelling herbivores, calling for body guards, and attracting pollinators.

Trifolium L., which belongs to the IR lacking clade (IRLC), is one of the largest genera in the Leguminosae and contains several economically important fodder species. Here, we present whole chloroplast (cp) genome sequencing and annotation of two important annual grasses, Trifolium alexandrinum (Egyptian clover) and T. resupinatum (Persian clover). Abundant single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels) were discovered between those two species. Global alignment of T. alexandrinum and T. resupinatum to a further thirteen Trifolium species revealed a large amount of rearrangement and repetitive events in these fifteen species. As hypothetical cp open reading frame (ORF) and RNA polymerase subunits, ycf1 and rpoC2 in the cp genomes both contain vast repetitive sequences and observed high Pi values (0.7008, 0.3982) between T. alexandrinum and T. resupinatum. Thus they could be considered as the candidate genes for phylogenetic analysis of Trifolium species. In addition, the divergence time of those IR lacking Trifolium species ranged from 84.8505 Mya to 4.7720 Mya. This study will provide insight into the evolution of Trifolium species.

To gain insight into the regulatory networks that underlie the induced defense in cucumber against spider mites, genes encoding transcription factors (TFs) were identified in the cucumber (Cucumissativus) genome and their regulation by two-spotted spider mite (Tetranychusurticae) herbivory was analyzed using RNA-seq. Of the total 1212 annotated TF genes in the cucumber genome, 119 were differentially regulated upon spider-mite herbivory during a period of 3 days. These TF genes belong to different categories but the MYB, bHLH, AP2/ERF and WRKY families had the highest relative numbers of differentially expressed genes. Correlation analysis of the expression of TF genes with defense-associated genes during herbivory and pathogen infestation, and in different organs resulted in the putative identification of regulators of herbivore-induced terpenoid and green-leaf-volatile biosynthesis. Analysis of the cis-acting regulatory elements (CAREs) present in the promoter regions of the genes responsive to spider-mite feeding revealed potential TF regulators. This study describes the TF genes in cucumber that are potentially involved in the regulation of induced defense against herbivory by spider mites.

The Cucurbitaceae family comprises economically valuable vegetables such as cucumber, melon, and pumpkin. GRAS proteins, which are crucial transcription factors, play diverse roles in plant growth and development. However, comparative investigations of GRAS proteins across Cucurbitaceae species are limited. Here, we identified 241 GRAS family genes in six cucurbit crops. The number of GRAS genes in cucumber, melon, wax gourd, watermelon, and bottle gourd ranged from 36 to 37, while the pumpkin genome contained 57 GRAS genes, possibly due to a recent whole-genome duplication. We classified cucurbit GRAS genes into 16 subfamilies and identified species-specific motifs and specific-expression patterns in the SCLB and RAD1 subfamilies. Notably, we identified 38 tissue-specific expressed genes, particularly fruit-specific genes potentially involved in fruit development. Additionally, we predicted the role of GRAS genes in regulating hypocotyl elongation under weak or dark light conditions in cucurbit plants. These findings enhance our understanding of the characteristics, evolution, and potential functions of GRAS genes in six cucurbit crops, providing valuable resources for genetic research in the Cucurbitaceae family as well as important agronomic traits.

Introduction: Citrus plants produce a rich diversity of volatile organic compounds (VOCs) that play important roles in fruit aroma, essential oils, and defense against biological stresses in aboveground tissues, yet volatile profiles in citrus roots remain poorly understood. Methods: Here, we employed an integrated metabolomic and transcriptomic approach to investigate VOCs and associated genes in Carrizo citrange ( Citrus sinensis × Poncirus trifoliata ) roots, by comparing control roots (CK), Rhizobium rhizogenes -induced hairy roots (HR) and those further elicited by methyl jasmonate (HR+MeJA). Results: We identified 642 VOCs in the root samples, with terpenoids representing the most dominant chemical class. Comparative analysis of differential metabolites and genes between HR, HR+MeJA, and controls revealed terpenoids and candidate genes that were responsive to R. rhizogenes induction and MeJA elicitation. Among them, a putative terpene synthase gene, Cs2g07240 , was found to be highly transcribed in several aboveground tissues and markedly upregulated upon Huanglongbing (HLB) infection. In vitro heterologous expression verified that this gene converted GPP to linalool and FPP to trans-nerolidol. However, overexpressing this gene in Carrizo citrange hairy roots promoted linalool and several non-target terpenes but not trans-nerolidol. Furthermore, stable overexpression of Cs2g07240 in sweet orange ( C. sinensis ) increased linalool accumulation in leaves, whereas trans-nerolidol remained undetectable. Leaves of the overexpression lines were also more attractive to adults of Diaphorina citri , the insect vector of HLB. Discussion: Collectively, this study unveils the terpenoid volatile repertoire in citrus roots and its modulation by R. rhizogenes and MeJA, while also highlighting the catalytic divergence of this synthase between in vitro and in planta contexts, as well as the tissue-specific complexity of terpenoid biosynthesis and ecological function.