High-Quality Assembly and Analysis of the Complete Mitogenomes of German Chamomile (Matricaria recutita) and Roman Chamomile (Chamaemelum nobile).

German chamomile (Matricaria chamomilla L.) and Roman chamomile (Chamaemelum nobile) are the two well-known chamomile species from the Asteraceae family. Owing to their essential oils and higher medicinal value, these have been cultivated widely across Europe, Northwest Asia, North America, and Africa. Regarding medicinal applications, German chamomile is the most commonly utilized variety and is frequently recognized as the "star among medicinal species". The insufficient availability of genomic resources may negatively impact the progression of chamomile industrialization. Chamomile's mitochondrial genome is lacking in extensive empirical research. In this study, we achieved the successful sequencing and assembly of the complete mitochondrial genome of M. chamomilla and C. nobile for the first time. An analysis was conducted on codon usage, sequence repeats within the mitochondrial genome of M. chamomilla and C. nobile. The phylogenetic analysis revealed a consistent positioning of M. chamomilla and C. nobile branches within both mitochondrial and plastid-sequence-based phylogenetic trees. Furthermore, the phylogenetic analysis also showed a close relationship between M. chamomilla and C. nobile within the clade comprising species from the Asteraceae family. The results of our analyses provide valuable resources for evolutionary research and molecular barcoding in chamomile.

Genome-Wide Identification of the IQM Gene Family and Their Transcriptional Responses to Abiotic Stresses in Kiwifruit (Actinidia eriantha).

IQM is a plant-specific calcium-binding protein that plays a pivotal role in various aspects of plant growth response to stressors. We investigated the IQM gene family and its expression patterns under diverse abiotic stresses and conducted a comprehensive analysis and characterization of the AeIQMs, including protein structure, genomic location, phylogenetic relationships, gene expression profiles, salt tolerance, and expression patterns of this gene family under different abiotic stresses. Based on phylogenetic analysis, these 10 AeIQMs were classified into three distinct subfamilies (I-III). Analysis of the protein motifs revealed a considerable level of conservation among these AeIQM proteins within their respective subfamilies in kiwifruit. The genomic distribution of the 10 AeIQM genes spanned across eight chromosomes, where four pairs of IQM gene duplicates were associated with segmental duplication events. qRT-PCR analysis revealed diverse expression patterns of these AeIQM genes under different hormone treatments, and most AeIQMs showed inducibility by salt stress. Further investigations indicated that overexpression of AeIQMs in yeast significantly enhanced salt tolerance. These findings suggest that AeIQM genes might be involved in hormonal signal transduction and response to abiotic stress in Actinidia eriantha. In summary, this study provides valuable insights into the physiological functions of IQMs in kiwifruit.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit.

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.

Genome-Wide Identification of HSF Gene Family in Kiwifruit and the Function of AeHSFA2b in Salt Tolerance.

Heat shock transcription factors (HSFs) play a crucial role in regulating plant growth and response to various abiotic stresses. In this study, we conducted a comprehensive analysis of the AeHSF gene family at genome-wide level in kiwifruit (Actinidia eriantha), focusing on their functions in the response to abiotic stresses. A total of 41 AeHSF genes were identified and categorized into three primary groups, namely, HSFA, HSFB, and HSFC. Further transcriptome analysis revealed that the expression of AeHSFA2b/2c and AeHSFB1c/1d/2c/3b was strongly induced by salt, which was confirmed by qRT-PCR assays. The overexpression of AeHSFA2b in Arabidopsis significantly improved the tolerance to salt stress by increasing AtRS5, AtGolS1 and AtGolS2 expression. Furthermore, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays demonstrated that AeHSFA2b could bind to the AeRFS4 promoter directly. Therefore, we speculated that AeHSFA2b may activate AeRFS4 expression by directly binding its promoter to enhance the kiwifruit's tolerance to salt stress. These results will provide a new insight into the evolutionary and functional mechanisms of AeHSF genes in kiwifruit.

Comparative Transcriptomic Analysis Revealed the Suppression and Alternative Splicing of Kiwifruit (Actinidia latifolia) NAP1 Gene Mediating Trichome Development.

Kiwifruit (Actinidia chinensis) is commonly covered by fruit hairs (trichomes) that affect kiwifruit popularity in the commercial market. However, it remains largely unknown which gene mediates trichome development in kiwifruit. In this study, we analyzed two kiwifruit species, A. eriantha (Ae) with long, straight, and bushy trichomes and A. latifolia (Al) with short, distorted, and spare trichomes, by second- and third-generation RNA sequencing. Transcriptomic analysis indicated that the expression of the NAP1 gene, a positive regulator of trichome development, was suppressed in Al compared with that in Ae. Additionally, the alternative splicing of AlNAP1 produced two short transcripts (AlNAP1-AS1 and AlNAP1-AS2) lacking multiple exons, in addition to a full-length transcript of AlNAP1-FL. The defects of trichome development (short and distorted trichome) in Arabidopsis nap1 mutant were rescued by AlNAP1-FL but not by AlNAP1-AS1. AlNAP1-FL gene does not affect trichome density in nap1 mutant. The qRT-PCR analysis indicated that the alternative splicing further reduces the level of functional transcripts. These results indicated that the short and distorted trichomes in Al might be caused by the suppression and alternative splicing of AlNAP1. Together, we revealed that AlNAP1 mediates trichome development and is a good candidate target for genetic modification of trichome length in kiwifruit.

A Comparative Genomics Approach for Analysis of Complete Mitogenomes of Five Actinidiaceae Plants.

Actinidiaceae, an economically important plant family, includes the Actinidia, Clematoclethra and Saurauia genus. Kiwifruit, with remarkably high vitamin C content, is an endemic species widely distributed in China with high economic value. Although many Actinidiaceae chloroplast genomes have been reported, few complete mitogenomes of Actinidiaceae have been studied. Here, complete circular mitogenomes of the four kiwifruit species and Saurauia tristyla were assembled. Codon usage, sequence repeats, RNA editing, gene transfers, selective pressure, and phylogenetic relationships in the four kiwifruit species and S. tristyla were comparatively analyzed. This research will contribute to the study of phylogenetic relationships within Actiniaceae and molecular barcoding in kiwifruit.

Genome-Wide Expression Profiling Analysis of Kiwifruit GolS and RFS Genes and Identification of AcRFS4 Function in Raffinose Accumulation.

The raffinose synthetase (RFS) and galactinol synthase (GolS) are two critical enzymes for raffinose biosynthesis, which play an important role in modulating plant growth and in response to a variety of biotic or abiotic stresses. Here, we comprehensively analyzed the RFS and GolS gene families and their involvement in abiotic and biotic stresses responses at the genome-wide scale in kiwifruit. A total of 22 GolS and 24 RFS genes were identified in Actinidia chinensis and Actinidia eriantha genomes. Phylogenetic analysis showed that the GolS and RFS genes were clustered into four and six groups, respectively. Transcriptomic analysis revealed that abiotic stresses strongly induced some crucial genes members including AcGolS1/2/4/8 and AcRFS2/4/8/11 and their expression levels were further confirmed by qRT-PCR. The GUS staining of AcRFS4Pro::GUS transgenic plants revealed that the transcriptionlevel of AcRFS4 was significantly increased by salt stress. Overexpression of AcRFS4 in Arabidopsis demonstrated that this gene enhanced the raffinose accumulation and the tolerance to salt stress. The co-expression networks analysis of hub transcription factors targeting key AcRFS4 genes indicated that there was a strong correlation between AcNAC30 and AcRFS4 expression under salt stress. Furthermore, the yeast one-hybrid assays showed that AcNAC30 could bind the AcRFS4 promoter directly. These results may provide insights into the evolutionary and functional mechanisms of GolS and RFS genes in kiwifruit.

EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in Arabidopsis.

The chloroplast is the main organelle for stress-induced production of reactive oxygen species (ROS). However, how chloroplastic ROS homeostasis is maintained under salt stress is largely unknown. We show that EGY3, a gene encoding a chloroplast-localized protein, is induced by salt and oxidative stresses. The loss of EGY3 function causes stress hypersensitivity while EGY3 overexpression increases the tolerance to both salt and chloroplastic oxidative stresses. EGY3 interacts with chloroplastic Cu/Zn-SOD2 (CSD2) and promotes CSD2 stability under stress conditions. In egy3-1 mutant plants, the stress-induced CSD2 degradation limits H2O2 production in chloroplasts and impairs H2O2-mediated retrograde signaling, as indicated by the decreased expression of retrograde-signal-responsive genes required for stress tolerance. Both exogenous application of H2O2 (or APX inhibitor) and CSD2 overexpression can rescue the salt-stress hypersensitivity of egy3-1 mutants. Our findings reveal that EGY3 enhances the tolerance to salt stress by promoting the CSD2 stability and H2O2-mediated chloroplastic retrograde signaling.

Cloning and functional analysis of three aphid alarm pheromone genes from German chamomile (Matricaria chamomilla L.).

German chamomile (Matricaria chamomilla L.) is one of the most ancient medicinal species in the world and terpenoids from their flowers have important medicinal value. We cloned three sesquiterpene synthase genes, McGDS1, McGDS2 and McGDS3, and performed sequence alignment and phylogenetic analysis. The encoded proteins possess three conserved structural features: an RRxxxxxxxxW motif, an RxR motif, and a DDxxD motif. McGDS1, McGDS2 and McGDS3 were confirmed to be (E)-farnesene synthase, germacrene D synthase, and germacrene A synthase, respectively. Subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus. qPCR indicated that McGDS1, McGDS2 and McGDS3, were more highly expressed in young flowers than in old flowers and the expression was highly correlated with amounts of the end-product essential oils ((E)-ß-farnesene, germacrene D and ß-elemene), with coefficients of 0.76, 0.83 and 0.68, respectively. We also established a transformation system for chamomile hairy roots. The overexpression of McGDS1, McGDS2 and McGDS3 resulted in γ-muurolene accumulation in hairy roots. The activity of three aphid alarm pheromones here forms the molecular basis for the study of the biosynthesis and regulation of volatile terpenes. Transformation of chamomile hairy roots provides a simple system in which to study terpene biosynthesis in chamomile.

Phytochemical and comparative transcriptome analyses reveal different regulatory mechanisms in the terpenoid biosynthesis pathways between Matricaria recutita L. and Chamaemelum nobile L.

BACKGROUND: Matricaria recutita (German chamomile) and Chamaemelum nobile (Roman chamomile) belong to the botanical family Asteraceae. These two herbs are not only morphologically distinguishable, but their secondary metabolites - especially the essential oils present in flowers are also different, especially the terpenoids. The aim of this project was to preliminarily identify regulatory mechanisms in the terpenoid biosynthetic pathways that differ between German and Roman chamomile by performing comparative transcriptomic and metabolomic analyses. RESULTS: We determined the content of essential oils in disk florets and ray florets in these two chamomile species, and found that the terpenoid content in flowers of German chamomile is greater than that of Roman chamomile. In addition, a comparative RNA-seq analysis of German and Roman chamomile showed that 54% of genes shared > 75% sequence identity between the two species. In particular, more highly expressed DEGs (differentially expressed genes) and TF (transcription factor) genes, different regulation of CYPs (cytochrome P450 enzymes), and rapid evolution of downstream genes in the terpenoid biosynthetic pathway of German chamomile could be the main reasons to explain the differences in the types and levels of terpenoid compounds in these two species. In addition, a phylogenetic tree constructed from single copy genes showed that German chamomile and Roman chamomile are closely related to Chrysanthemum nankingense. CONCLUSION: This work provides the first insights into terpenoid biosynthesis in two species of chamomile. The candidate unigenes related to terpenoid biosynthesis will be important in molecular breeding approaches to modulate the essential oil composition of Matricaria recutita and Chamaemelum nobile.

Analysis of terpenoid biosynthesis pathways in German chamomile (Matricaria recutita) and Roman chamomile (Chamaemelum nobile) based on co-expression networks.

German chamomile and Roman chamomile are the two most widely known chamomile species due to the medicinal properties of volatile compounds from their flowers. We determined the volatile compound content of different organs of these two chamomiles, and found that main volatile compounds in German chamomile were terpenoids and those in Roman chamomile were esters. Furthermore, 24 tissues from two chamomiles were sequenced and analyzed by gene co-expression network. The results showed higher terpene synthase expression levels and more modules correlated with sesquiterpenoids in German chamomile, which may explain its high sesquiterpenoid content. In both chamomiles, unigenes in volatile compound-correlated modules were significantly enriched in pathways related to plant-pathogen interactions and circadian rhythm, demonstrating that volatile compounds of chamomiles are influenced by these factors. There were ten times more unigenes related to plant-pathogen interactions in German chamomile than in Roman chamomile, which indicates German chamomile has higher resistance to pathogens.

Comparative Transcriptomic Analysis Reveals Regulatory Mechanisms of Theanine Synthesis in Tea (Camellia sinensis) and Oil Tea (Camellia oleifera) Plants.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (Camellia sinensis) and oil tea (Camellia oleifera) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.