Cis-acting Research Articles

Characterization of Superoxide Dismutase (SOD) Gene Family and Their Responses to Salinity Stress and Fruit Development in Octoploid Strawberry

Superoxide dismutases (SODs), as the first line of defense against reactive oxygen species (ROS), play an essential role in protecting plants from adverse elicitors during plant growth and development. However, little is known about the SOD gene family and their response to salinity stress and fruit development in cultivated strawberries (Fragaria × ananassa). In this study, 32 SOD genes consisting of 16 Cu/ZnSODs, 11 FeSODs, and 5 MnSOD were identified, which presented three well-resolved clades in the phylogenetic tree. Each clade had similar motifs, and exon–intron structures, which in turn supported the evolutionary classification. Cis-acting element analysis suggested that FaSOD genes might be involved in the plant response to abiotic and biotic stresses, hormones, and light. The analysis of previously published transcriptome data revealed that FaSOD genes are expressed variably under salt stress. Among these SODs, FaMSD5 was expressed at relatively high levels in strawberry root and leaf, and its transcript abundance significantly increased after salt treatment. Some transcription factors related to photomorphogenesis, hormone signaling pathways, and hyperosmotic salinity response were predicted to bind to the FaMSD5 promoter. These outcomes implied that FaMSD5 might play an important role in protection against salt stress. In addition, the comprehensive transcriptome analysis of FaSOD genes in strawberry fruit showed that almost all FaCSDs and FaMSDs were more highly expressed than FaFSDs at different developmental stages, and the expression patterns of FaCSD1, FaCSD2, FaCSD7, FaCSD8, and FaCSD10 suggested that they were likely to be involved in fruit development and ripening. This study provides a basis for further exploration of the function of the FaSOD gene family in strawberry and provides candidate FaSOD genes for enhancing salinity tolerance and regulating fruit development and ripening.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Genome-wide computational analysis of the dirigent gene family in Solanum lycopersicum

BackgroundDirigent (DIR) genes play a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. They are required to produce lignans, which are a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential role in plant development. DIR genes are implicated in the process of lignification and protect plants from environmental stresses, including biotic and abiotic stresses. Nevertheless, no research has been performed on the DIR gene family in Solanum lycopersicum. This study provides detailed information on the DIR gene family in S. lycopersicum.Methods and resultsThe conserved domain analysis, phylogenetic analysis, evolutionary adaptation, cis-acting elements, proteomic analysis, signal peptide detection, transmembrane potential analysis, sequence identity and similarity analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of 31 potential DIR genes were studied. All these analyses provide a deep understanding of DIR genes in the S. lycopersicum genome that will provide a useful reference for further functional analysis of the DIR genes in S. lycopersicum.ConclusionThis research provides an in-depth and comprehensive explanation of the detailed process and structural characterization of DIR genes in the genome of S. lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This work will provide valuable information for identifying DIR genes in higher plants and support future research on the DIR gene family.

Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa

Plant-specific APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factors are involved in the regulation of genes associated with the growth and developmental processes of numerous plants. Although AP2/ERF proteins from other species have been intensively studied, no studies have been reported on the AP2/ERF family of Luffa cylindrica, an important vegetable of the cucurbit family, and one of the most popular vegetables in the world. In this study, 133 genes (315–6696 bp) encoding LcAP2/ERF proteins with complete AP2/ERF domains were identified according to the luffa P93075 genome. These LcAP2/ERF genes were subsequently classified and analyzed for their gene structures, chromosomal distribution locations, promoter cis-acting elements, conserved structural domains of encoded proteins, and responses to abiotic stresses. The LcAP2/ERF genes were identified and divided into five phylogenetic groups (AP2, DREBs, ERFs, RAV, and soloists). These genes were unevenly distributed across 13 chromosomes. An analysis of gene structures indicated the LcAP2/ERF genes contained 0–11 introns (average of 4.4). Additionally, 16 motifs were identified in the LcAP2/ERF proteins that were conserved across different phylogenetic groups. Moreover, 11 cis-acting elements associated with response to the environment were analyzed in a 2000 bp region upstream of the LcAP2/ERF gene promoters. A transcriptome analysis involving RNA-seq data revealed tissue-specific LcAP2/ERF expression profiles and the diversity in LcAP2/ERF expression. The effects of low-temperature stress on LcAP2/ERF expression were determined. Furthermore, fruit-development-related and low-temperature-induced expressional changes were verified by RT-qPCR analyses of 14 differentially expressed LcAP2/ERF genes in luffa. Our findings will help clarify the evolution of the luffa AP2/ERF family, while also providing valuable insights for future studies on AP2/ERF functions.

Terpene synthase gene family in Jasminum sambac var. Fuzhou bifoliatum: genome-wide analysis and expression pattern in response to methyl jasmonate

Terpene synthases (TPSs) play a crucial role in the synthesis of terpenoids that contribute to the scent profiles of flowers. However, few studies report the genome-wide analysis of TPSs gene in Jasminum sambac var. Fuzhou bifoliatum and their expression pattern in response to methyl jasmonate (MeJA). In this study, we employed bioinformatics tools for genome-wide analysis of the J. sambac TPS (DJTPS) gene family and determined the physical and chemical properties, subcellular location, protein-protein interactions, phylogenetic relationship, subfamily classification, chromosomal location and collinearity, gene structure, conserved motifs, and promoter cis-acting elements. The expression patterns of DJTPSs in different tissues and in response to MeJA treatment were analyzed based on the transcriptome data combined with quantitative real-time PCR (qRT-PCR). We identified 32 intact DJTPS genes in the genome of J. sambac, which presented uneven distribution across nine chromosomes. All the deduced proteins were hydrophilic, predominantly localized in the cytoplasm. The phylogenetic analysis classified the DJTPS genes into five subfamilies: TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. The results of the collinearity analysis showed a total of 10 sets of replication events in DJTPSs, most of which underwent purifying selection. A comparative analysis of TPS homologous gene pairs was performed among J. sambac var. Fuzhou bifoliatum and other six species, which revealed different number of homologous gene pairs. The number of exons and motifs was conserved within the same subfamily. DJTPS genes carried multiple elements that may be involved in the response to MeJA. In addition, the transcriptome and qRT-PCR data unveiled that several TPS genes exhibited tissue-specific expression patterns, and the genes with specific expression in flowers were the most. Upon exposure to MeJA, 14 TPS genes showcased upregulated expression 5 h or 6 h post-treatment, and DJTPS03, DJTPS04 and DJTPS21 showed significantly increased expression levels after MeJA treatment. This study provides preliminary evidence that MeJA possesses the ability to enhance the expression of DJTPS genes during the critical flowering stage, which will facilitate the synthesis of terpenoids and improve the quality of floral fragrance.

Genome-wide analysis of the SWEET gene family and its response to powdery mildew and leaf spot infection in the common oat (Avena sativa L.)

The nutritional quality and yield of oats (Avena sativa) are often compromised by plant diseases such as red leaf, powdery mildew, and leaf spot. Sugars Will Eventually be Exported Transporters (SWEETs) are newly identified sugar transporters involved in regulating plant growth and stress responses. However, the roles of SWEET genes in biotic stress responses remain uncharacterized in oats. In this study, 13 AsSWEET genes were identified across nine chromosomes of the oat genome, all of which were predicted to contain seven transmembrane regions. Phylogenetic analysis revealed four clades of AsSWEET proteins, with high homology to SWEET proteins in the Poaceae family. Collinearity analysis demonstrated strong relationships between oat and Zea mays SWEETs. Using subcellular localization prediction tools, AsSWEET proteins were predicted to localize to the plasma membrane. Promoter analysis revealed cis-acting elements associated with light response, growth, and stress regulation. Six AsSWEET proteins were predicted to interact in a network centered on AsSWEET1a and AsSWEET11. Gene expression analysis of two oat varieties, ‘ForagePlus’ and ‘Molasses’, indicated significant expression differences in several AsSWEET genes following infection with powdery mildew or leaf spot, including AsSWEET1a, AsSWEET1b, AsSWEET2b, AsSWEET3a, AsSWEET11, and AsSWEET16. These SWEET genes are potential candidates for disease resistance in oats. This study provides a foundation for understanding the regulatory mechanisms of AsSWEET genes, particularly in response to powdery mildew and leaf spot, and offers insights for enhancing oat molecular breeding.

Machine-guided design of cell-type-targeting cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1-3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4-8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.

Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.)

Walnut (Juglans regia L.) possesses the ability to prevent coronary heart disease and promote cardiovascular health. This ability can be attributed to their rich content of polyphenols, particularly flavonoids. The biosynthesis of flavonoids is reliant on the catalytic activity of uridine diphosphate glycosyltransferase (UGT). However, the identification of UGTs in walnut has not been reported. In the current study, a total of 124 UGT genes containing the PSPG box were identified from the walnut genome. Based on phylogenetic analysis, the 124 UGTs could be classified into 16 distinct groups, which exhibited an uneven distribution across the 16 chromosomes. Subcellular localization prediction analysis revealed that approximately 78.23% of walnut UGT proteins were predominantly localized in the cytoplasmic compartment. Furthermore, motif annotation confirmed that motifs 1, 2, and 3 represented conserved structural features within UGT proteins, while interestingly, around 56.5% of walnut UGT members lacked introns. Through the analysis of promoter cis-regulatory elements, it was revealed that JrUGTs are involved in photoresponse, hormonal regulation, and other physiological responses. In conjunction with transcriptome analysis and quantitative expression, approximately 39% of UGT genes in walnut exhibited high expression levels during early fruit development. Correlation analysis between UGT genes’ expression and phenolic content in walnut indicated that JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 might be involved in phenolic biosynthesis in walnut. This comprehensive study provides an overview of the UGT genes in walnut, serving as a valuable reference and theoretical foundation for further investigations into the biological functions of JrUGTs in flavonoid biosynthesis.

Genome-Wide Identification of COMT Gene Family in Maize and its Function in Response to Light.

Maize is a major crop, feed, and industrial material. Caffeic acid-O-methyltransferase (COMT) is a methylase closely associated with lignin biosynthesis and plant growth and resistance. In this study, we identified the COMT gene (ZmCOMT) family in maize and further analyzed its phylogenetic evolution, subcellular localization, and its function in response to light. Thirty-one ZmCOMT genes were identified in the maize genome, which were distributed across eight chromosomes and mainly clustered on chromosome 4. Most ZmCOMT proteins were predicted to localize in the cytoplasm. Ten different conserved motifs were present in most ZmCOMT proteins, and motif1, motif6, and motif7 were highly conserved and present in all ZmCOMT proteins. The photoresponsivity elements were conserved among all members, and ZmCOMT22 and ZmCOMT10 genes responsive to light. This result suggests a potential function for these two genes in lignin biosynthesis which a previous study had linked to light regulation. Jasmonic acid responsive and abscisic acid cis-acting elements were present in the promoter regions of family members, thus the family may be regulated by hormone signaling pathways of maize. In summary, ZmCOMT genes are ancient, and the highly conserved motifs may be significant in survival and evolution of maize. Furthermore, light may influence lignin biosynthesis and photosynthesis through ZmCOMT genes. This research provided theoretical basis for lignin biosynthesis of maize and the potential value of ZmCOMT22 and ZmCOMT10 genes to enhance plant photosynthesis for facing global warming.

Genome Analysis of BnCNGC Gene Family and Function Exploration of BnCNGC57 in Brassica napus L.

The cyclic nucleotide-gated ion channel (CNGC), as a non-selective cation channel, plays a pivotal role in plant growth and stress response. A systematic analysis and identification of the BnCNGC gene family in Brassica napus is crucial for uncovering its biological functions and potential applications in plant science. In this study, we identified 61 BnCNGC members in the B. napus genome, which are phylogenetically similar to Arabidopsis and can be classified into Groups I-IV (with Group IV further subdivided into IV-a and IV-b). Collinearity analysis with other species provided insights into the evolution of BnCNGC. By homology modeling, we predicted the three-dimensional structure of BnCNGC proteins and analyzed cis-acting elements in their promoters, revealing diverse roles in hormone regulation, growth, and stress response. Notably, overexpression of BnCNGC57 (BnaC09g42460D) significantly increased seed size, possibly through regulating cell proliferation via the MAPK signaling pathway. Our findings contribute to a better understanding of the BnCNGC gene family and highlight the potential regulatory role of BnCNGC57 in the seed development of B. napus.

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.

The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

Genome-wide analysis and expression profile of the bZIP gene family in Neopyropia yezoensis.

The basic leucine zipper (bZIP) family consists of conserved transcription factors which are widely present in eukaryotes and play important regulatory roles in plant growth, development, and stress responses. Neopyropia yezoensis is a red marine macroalga of significant economic importance; however, their bZIP family members and functions have not been systematically identified and analyzed. In the present study, the bZIP gene family in Ny. yezoensis was characterized by investigating gene structures, conserved motifs, phylogenetic relationships, chromosomal localizations, gene duplication events, cis-regulatory elements, and expression profiles. Twenty-three Ny. yezoensis bZIP (NyybZIP) genes were identified and sorted into 13 out of 30 groups, which were classified based on the bZIPs of Ny. yezoensis and 15 other red algae species. Phylogenetic analysis revealed that bZIP genes may have a complex evolutionary pattern in red algae. Cross-species collinearity analysis indicated that the bZIP genes in Ny. yezoensis, Neoporphyra haitanensis, and Porphyra umbilicalis are highly evolutionarily conserved. In addition, we identified four main categories of cis-elements, including development-related, light-responsive, phytohormone-responsive and stress-responsive promoter sequences in NyybZIP genes. Finally, RNA sequencing data and quantitative real-time PCR (qRT-PCR) showed that NyybZIP genes exhibited different expression patterns depending on the life stage. NyybZIP genes were also found to be involved in the nitrogen stress response. We thought that bZIP genes may be involved in Ny. yezoensis growth and development, and play a significant role in nitrogen deficiency response. Taken together, our findings provide new insights into the roles of the bZIP gene family and provide a basis for additional research into its evolutionary history and biological functions.

Genome-Wide Identification of the Cyclic Nucleotide-Gated Ion Channel Gene Family and Expression Profiles Under Low-Temperature Stress in Luffa cylindrica L.

Cyclic nucleotide-gated ion channels (CNGCs) are cell membrane channel proteins for calcium ions. They have been reported to play important roles in survival and in the responses to environmental factors in various plants. However, little is known about the CNGC family and its functions in luffa (Luffa cylindrica L.). In this study, a bioinformatics-based method was used to identify members of the CNGC gene family in L. cylindrica. In total, 20 LcCNGCs were detected, and they were grouped into five subfamilies (I, II, Ⅲ, IV-a, and IV-b) in a phylogenetic analysis with CNGCs from Arabidopsis thaliana (20 AtCNGCs) and Momordica charantia (17 McCNGCs). The 20 LcCNGC genes were unevenly distributed on 11 of the 13 chromosomes in luffa, with none on Chromosomes 1 and 5. The members of each subfamily encoded proteins with highly conserved functional domains. An evolutionary analysis of CNGCs in luffa revealed three gene losses and a motif deletion. An examination of gene replication events during evolution indicated that two tandemly duplicated gene pairs were the primary driving force behind the evolution of the LcCNGC gene family. PlantCARE analyses of the LcCNGC promoter regions revealed various cis-regulatory elements, including those responsive to plant hormones (abscisic acid, methyl jasmonate, and salicylic acid) and abiotic stresses (light, drought, and low temperature). The presence of these cis-acting elements suggested that the encoded CNGC proteins may be involved in stress responses, as well as growth and development. Transcriptome sequencing (RNA-seq) analyses revealed tissue-specific expression patterns of LcCNGCs in various plant parts (roots, stems, leaves, flowers, and fruit) and the upregulation of some LcCNGCs under low-temperature stress. To confirm the accuracy of the RNA-seq data, 10 cold-responsive LcCNGC genes were selected for verification by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Under cold conditions, LcCNGC4 was highly upregulated (>50-fold increase in its transcript levels), and LcCNGC3, LcCNGC6, and LcCNGC13 were upregulated approximately 10-fold. Our findings provide new information about the evolution of the CNGC family in L. cylindrica and provide insights into the functions of the encoded CNGC proteins.

Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species

Auxin is the first plant hormone found to play a dominant role in fruit growth, from fruit set to fruit ripening. Strawberry plants represent a suitable model for studying auxin’s biosynthesis, sensing, and signaling machinery. Aux/IAA genes are a classical rapid auxin-responsive family. However, the Aux/IAA gene family in Fragaria genus is poorly understood. In this study, a total of 287 Aux/IAA genes were identified in the eight strawberry genomes. Their physicochemical properties, domain structure, and cis-regulatory elements revealed the functional multiplicity of the strawberry Aux/IAAs. We used a phylogenetic analysis to classify these genes into 12 classes. In addition, based on synteny analysis, gene duplications, and calculation of the Ka/Ks ratio, we found that segmental duplications promote the evolution of Aux/IAAs in Fragaria species, which is followed by purifying selection. Furthermore, the expression pattern and protein–protein interaction network of these genes in Fragaria vesca revealed various tissue-specific expressions and probable regulatory functions. Taken together, these results provide basic genomic information and a functional analysis of these genes, which will serve to expand our understanding of the direction in which the Aux/IAA gene family is evolving in Fragaria species.

GhASHH1.A and GhASHH2.A Improve Tolerance to High and Low Temperatures and Accelerate the Flowering Response to Temperature in Upland Cotton (Gossypium hirsutum).

The trithorax group (TrxG) complex is an important protein in the regulation of plant histone methylation. The ABSENT, SMALL, OR HOMEOTIC DISCS 1 (ASH1) gene family, as important family members of the TrxG complex, has been shown to regulate tolerance to abiotic stress and growth and development in many plants. In this study, we identified nine GhASH1s in upland cotton. Bioinformatics analysis revealed that GhASH1s contain a variety of cis-acting elements related to stress resistance and growth and development. The transcriptome expression profiles revealed that GhASHH1.A and GhASHH2.A genes expression were upregulated in flower organs and in response to external temperature stress. The results of virus-induced gene silencing (VIGS) indicated that GhASHH1.A and GhASHH2.A genes silencing reduced the ability of cotton to adapt to temperature stress and delayed the development of the flowering phenotype. We also showed that the silencing of these two target genes did not induce early flowering at high temperature (32 °C), suggesting that GhASHH1.A and GhASHH2.A might regulate cotton flowering in response to temperature. These findings provide genetic resources for future breeding of early-maturing and temperature-stress-tolerant cotton varieties.

Genome-Wide Analysis and Expression Profiling of Glyoxalase Gene Families Under Abiotic Stresses in Cucumber (Cucumis sativus L.)

The glyoxalase pathway, consisting of glyoxalase I (GLYI) and glyoxalase II (GLYII), is an enzymatic system that converts cytotoxic methylglyoxal to non-toxic S-D-lactoylglutathione. Although the GLY gene family has been analyzed in Arabidopsis, rice, grape, cabbage, and soybean, cucumber studies are lacking. Here, we analyzed the cucumber GLY gene family, identifying 13 CsGLYI and 2 CsGLYII genes. Furthermore, we investigated the physicochemical properties, phylogenetic relationships, chromosomal localization and colinearity, gene structure, conserved motifs, cis-regulatory elements, and protein–protein interaction networks of the CsGLY family. They were primarily localized in the cytoplasm, chloroplasts, and mitochondria, with a minor presence in the nucleus. The classification of CsGLYI and CsGLYII genes into five classes closely resembled the homologous genes in Arabidopsis and soybean. Additionally, hormone-responsive elements dominated the promoter region of GLY genes, alongside light- and stress-responsive elements. The predicted interaction proteins of CsGLYIs and CsGLYIIs exerted a significant role in cellular respiration, amino acid synthesis, and metabolism, as well as methylglyoxal catabolism. In addition, the expression profiles of GLY genes were distinct in different tissues of cucumber as well as under diverse abiotic stresses. This study is conducive to the further exploration of the functional diversity among glyoxalase genes and the mechanisms of stress responses in cucumber.

A single dominant GLOBOSA allele accounts for repeated origins of hose-in-hose flowers in Sinningia (Gesneriaceae).

Plants bearing double flowers have long been cultivated as ornamental plants. Hose-in-hose flowers, bearing 2-whorled corolla tubes in whorls 1 and 2, are uncommon but recur in Sinningia (Gesnerioideae, Gesneriaceae). In this study, we selected 15 hose-in-hose cultivars as materials to explore the underlying molecular and genetic mechanisms of this floral architecture. We found that they originated from different hybridization events within the Dircaea clade. Three B-class MADS-box genes were globally expressed in all floral whorls, but only GLOBOSA1 (GLO1) has accumulated a dominant mutation, i.e., the insertion of a hAT-like miniature inverted-repeat transposable element (MITE) into its promoter, that co-segregated with the hose-in-hose phenotype. In addition, all 15 hose-in-hose cultivars contained the same dominant GLO1 allele. Transient gene expression assays confirmed the role of this MITE insertion in up-regulating the promoter activity of GLO1 by providing several cis-regulatory elements. Genetic transformation in heterologous Chirita pumila (Didymocarpoideae, Gesneriaceae) verified that this dominant GLO1 allele is sufficient to confer the hose-in-hose phenotype. We further demonstrated that both the GLO1 allele and the hAT-like MITE descended from wild S. cardinalis with single flowers. This study highlights the significance of wide hybridization in frequent gains of the dominant GLO1 allele and thereafter repeated occurrence of hose-in-hose flowers in Sinningia.

A novel interpretable deep learning-based computational framework designed synthetic enhancers with broad cross-species activity.

Enhancers play a critical role in dynamically regulating spatial-temporal gene expression and establishing cell identity, underscoring the significance of designing them with specific properties for applications in biosynthetic engineering and gene therapy. Despite numerous high-throughput methods facilitating genome-wide enhancer identification, deciphering the sequence determinants of their activity remains challenging. Here, we present the DREAM (DNA cis-Regulatory Elements with controllable Activity design platforM) framework, a novel deep learning-based approach for synthetic enhancer design. Proficient in uncovering subtle and intricate patterns within extensive enhancer screening data, DREAM achieves cutting-edge sequence-based enhancer activity prediction and highlights critical sequence features implicating strong enhancer activity. Leveraging DREAM, we have engineered enhancers that surpass the potency of the strongest enhancer within the Drosophila genome by approximately 3.6-fold. Remarkably, these synthetic enhancers exhibited conserved functionality across species that have diverged more than billion years, indicating that DREAM was able to learn highly conserved enhancer regulatory grammar. Additionally, we designed silencers and cell line-specific enhancers using DREAM, demonstrating its versatility. Overall, our study not only introduces an interpretable approach for enhancer design but also lays out a general framework applicable to the design of other types of cis-regulatory elements.

Cloning and Expression Analysis of ATG8 (Autophagy-Related 8) Gene Family in Solanaceae.

The autophagy-related gene family ATG8 (Autophagy-related 8) plays an important role in plant growth, development, and stress response. In this study, 15 ATG8 gene family sequences were amplified from Solanaceae, namely tobacco, tomato, and pepper, using RT-PCR to evaluate their basic properties, protein structure, and function, as well as the role of ATG8 in autophagy. The physicochemical properties, the predicted secondary and tertiary protein structures, subcellular localisation, gene structures, conserved motifs, and phylogenetic relationships of the ATG8 genes were analysed using bioinformatic techniques, and their expression patterns under sericin-induced plant disease resistance were investigated by RT-qPCR. The lengths of these proteins ranged from 79 to 120 aa, while their predicted molecular weights and isoelectric points (PI) ranged from 9283.62 to 13,778.74 and 6.32 to 11.44, respectively. The majority of the proteins were localised in the nucleus or chloroplasts. Conserved protein motifs and various cis-regulatory elements in the protein, with a wide range of related functions, were identified. The ATG8 gene family members showed expression changes after treatment with osthole, which induces disease resistance in tobacco, tomato, and pepper. These findings provide a foundation for further analyses of the ATG8 gene family in Solanaceae and the mechanism underlying the response to adverse conditions.

Genome-Wide Identification of MKK Gene Family and Response to Hormone and Abiotic Stress in Rice.

Mitogen-activated protein kinase (MAPK/MPK) cascades are pivotal and highly conserved signaling modules widely distributed in eukaryotes; they play essential roles in plant growth and development, as well as biotic and abiotic stress responses. With the development of sequencing technology, the complete genome assembly of rice without gaps, T2T (Telomere-to-Telomere)-NIP (version AGIS-1.0), has recently been released. In this study, we used bioinformatic approaches to identify and analyze the rice MPK kinases (MKKs) based on the complete genome. A total of seven OsMKKs were identified, and their physical and chemical properties, chromosome localization, gene structure, subcellular localization, phylogeny, family evolution, and cis-acting elements were evaluated. OsMKKs can be divided into four subgroups based on phylogenetic relationships, and the family members located in the same evolutionary branch have relatively similar gene structures and conserved domains. Quantitative real-time PCR (qRT-PCR) revealed that all OsMKKs were highly expressed in rice seedling leaves. The expression levels of all OsMKKs were more or less altered under exogenous hormone and abiotic stress treatments, with OsMKK1, OsMKK6, and OsMKK3 being induced under almost all treatments, while the expression of OsMKK4 and OsMKK10-2 was repressed under salt and drought treatments and IAA treatment, respectively. In this study, we also summarized the recent progress in rice MPK cascades, highlighted their diverse functions, and outlined the potential MPK signaling network, facilitating further studies on OsMKK genes and rice MPK cascades.