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Insect phenoloxidase, presented as an inactive precursor prophenoloxidase (PPO) in hemolymph, catalyzes melanin formation, which is involved in wound healing, pathogen killing, reversible oxygen collection during insect respiration, and cuticle and eggshell formation. Mosquitoes possess 9 to 16 PPO members across different genera, a number that is more than that found in other dipteran insects. However, the reasons for the redundancy of these PPOs and whether they have distinct biochemical properties and physiological functions remain unclear. Phylogenetic analysis confirmed that Aedes aegypti PPO6 (Aea-PPO6) is an ortholog to PPOs in other insect species, classified as the classical insect type, while other Aea-PPOs are unique to Diptera, herein referred to as the dipteran type here. We characterized two Aea-PPO members, Aea-PPO6, the classical insect type, and Aea-PPO10, a dipteran type, which exhibit distinct substrate specificities. By resolving Aea-PPO6' s crystal structure and creating a chimera protein (Aea-PPO6-cm) with Motif 1 (217GDGPDSVVR225) from Aea-PPO10, we identified the motif that determines PPO substrate specificity. In vivo, loss of Aea-PPO6 led to larval lethality, while Aea-PPO10 was involved in development, pigmentation, and immunity. Our results enhance the understanding of the functional diversification of mosquito PPOs. [ABSTRACT FROM AUTHOR]

Potassium, an essential inorganic cation, is crucial for the growth of oil crops like Brassica napus L. Given the scarcity of potassium in soil, enhancing rapeseed's potassium utilization efficiency is of significant importance. This study identified 376 potassium utilization genes in the genome of B. napus ZS11 through homologous retrieval, encompassing 7 functional and 12 regulatory gene families. These genes are unevenly distributed across 19 chromosomes, and the proteins encoded by these genes are mainly localized in the cell membrane, vacuoles, and nucleus. Microsynteny analysis highlighted the role of small-scale replication events and allopolyploidization in the expansion of potassium utilization genes, identifying 77 distinct types of cis-acting elements within their promoter regions. The regulatory mechanisms of potassium utilization genes were provided by analyses of transcription factors, miRNA, and protein interaction networks. Under low potassium stress, the potassium utilization genes, particularly those belonging to the KUP and CBL families, demonstrate pronounced co-expression. RNA-seq and RT-qPCR analysis identified the BnaZSHAK5.2 gene, which is a high-affinity potassium ion transporter, playing a crucial role in the stress response to potassium deficiency in B. napus, as its expression is strongly induced by low potassium stress. A functional complementation study demonstrates that the BnaZSHAK5.2 gene could rescue the primary root growth of the Athak5 mutant under low potassium conditions, confirming its role in response to low potassium stress by sustaining root development. [ABSTRACT FROM AUTHOR]

Simple Summary: Bactrocera dorsalis (Hendel), known as oriental fruit fly, is a global highly invasive insect species. We found that the posterior thoracic scutella of some B. dorsalis adults are yellow, some light yellow, and some white in the citrus orchards of China. In this study, we explored the molecular and biochemical mechanisms of scutellum color variation in B. dorsalis adults. KEGG enrichment analysis showed that genes associated with scutellum color variation were mainly enriched in these pathways of oxidative phosphorylation, porphyrin and chlorophyll metabolism, and terpenoid backbone biosynthesis. Biochemical analysis showed that β-carotene was one of the main pigments causing the B. dorsalis scutella to appear yellow. Bactrocera dorsalis (Hendel) is an invasive fruit and vegetable pest, infesting citrus, mango, carambola, etc. We observed that the posterior thoracic scutella of some B. dorsalis adults are yellow, some light yellow, and some white in China. Compared with the B. dorsalis races with a yellow scutellum (YS) and white scutellum (WS), the race with a light-yellow scutellum (LYS) is dominant in citrus and carambola orchards. To reveal genetic correlates among the three races, the genomes of 22 samples (8 with YS, 7 with LYS, and 7 with WS) were sequenced by high-throughput sequencing technology. Single-nucleotide polymorphism (SNP) annotation showed that there were 17,580 non-synonymous mutation sites located in the exonic region. Principal component analysis based on independent SNP data revealed that the SNPs with LYS were more similar to that with YS when compared with WS. Most genes associated with scutellum color variation were involved in three pathways: oxidative phosphorylation, porphyrin and chlorophyll metabolism, and terpenoid backbone biosynthesis. By comparing the sequences among the three races, we screened out 276 differential genes (DGs) in YS vs. WS, 185 DGs in LYS vs. WS, and 104 DGs in YS vs. LYS. Most genes determining color variation in B. dorsalis scutella were located on chromosomes 2–5. Biochemical analysis showed that β-carotene content in YS and LYS was significantly higher than that in WS at any stage of adult days 1, 10, and 20. No significant differences were observed in cytochrome P450 or melanin content in YS, LYS, or WS. Our study provides results on aspects of scutellum color variation in B. dorsalis adults, providing molecular and physiological information for revealing the adaptation and evolution of the B. dorsalis population. [ABSTRACT FROM AUTHOR]

While the rapid rise in bioinformatics has facilitated the identification of the domains and functions of many proteins, some still have no domain annotation or largely uncharacterized functions. However, the biological roles of unknown proteins were not clear in oomycetes. An analysis of the Phytophthora sojae genome database identified the protein Ps495620, which has no domain annotations and functional predictions in Phytophthora. This study used a CRISPR/Cas9-mediated gene replacement system to knock out Ps495620 to elucidate its function. The Ps495620-knockout mutants exhibited significantly increased oospore production and decreased sporangium formation compared to the wild-type strain P6497. Transcriptomics showed that it is a key regulator of nitrogen, pyruvate, ascorbate, and adorate metabolism in P. sojae. Our findings indicate that Ps495620 is critical in regulating sporangium formation and oospore production in P. sojae. [ABSTRACT FROM AUTHOR]

Citric acid is an important organic acid with wide applications and diverse biological functionality. As the predominant organic acid in lemons, citric acid plays a crucial role in determining the flavor of citrus, especially in lemons. ATP citrate lyase (ACL, EC4.1.3.8) is the keg gene in citric acid metabolism. Several research studies on ACL only focused on high-sugar- and low-acid-content citrus varieties; however, the ACL mechanism in lemons with high acid and low sugar levels remains undetermined. In this study, a key candidate gene, ClACLB-1, for citrate cleavage was identified from the genome data of 'Xiangshui' lemon [Citrus limon (L.) Burm f.]. The putative protein coded by the gene ClACLB-1 is localized in the nuclear and cell membranes. The ClACLB-1 gene was expressed in all tissues, with the highest expression in male flowers and the lowest expression in mature fruits; the expression decreased during lemon fruit development. The overexpression of ClACLB-1 in transgenic tomatoes significantly increases the activity of citrate lyase, which subsequently reduces citric acid content. This study clarified the function of the ClACLB-1 gene in cleaving citric acid, provided new insights into the citric acid metabolism of citrus, and offered a theoretical reference for reducing acid and increasing sugar in citrus to improve fruit quality. It also helped to enhance the understanding of the metabolism and role of citrate in plants. [ABSTRACT FROM AUTHOR]

KAR (Karrikin), a novel plant growth regulator, can be recognized specifically by plants and can activate resistance responses. MdKAI2 is the natural receptor of KARs in apple. Here, we report the identification of osmotic stress resistance in MdKAI2 via the method of genetic transformation. The phenotypic traits, resistance indicators, and transcriptional and metabolic regulation of MdKAI2 were identified. KAR1, a highly active form of KAR, markedly promoted the root growth of Gala cultivar tissue culture‒generated plants, possibly through increases in ABA and TZR contents and decreases in the GA3 content. MdKAI2 was markedly upregulated by PEG stress and significantly promoted the growth of apple calli under nonstress conditions, whereas it was significantly inhibited under 20% PEG stress, as was cell death. MdKAI2 significantly increased the content of total flavonoids, the activity of reactive oxygen species (ROS)‒scavenging enzymes (SOD, POD and CAT), and the content of osmoregulatory substances (soluble protein, soluble sugars and proline). It also inhibited the MDA content and conductivity under osmotic stress. Differentially expressed genes (DEGs), including multiple transcription factors (TFs), such as MYB , bHLH and AP2‒EREBP , are significantly regulated by MdKAI2, and genes involved in the mitogen‒activated protein kinase (MAPK) signaling pathway play crucial roles in the regulation of plant resistance. In addition, pathways such as brassinosteroid (BR) biosynthesis and ABC transporters were downregulated, and the MAPK signaling pathway; plant‒pathogen interaction; cutin, suberin and wax biosynthesis; alpha‒linolenic acid metabolism; and phenylpropanoid biosynthesis were upregulated by MdKAI2. MdKAI2 significantly regulates the levels of lipids, amino acids, terpenoids, benzene, organic acids, carbohydrates, and alkaloids and is involved in the metabolic processes of amino acids, carbohydrates, nucleotides, lipids and secondary metabolites. Furthermore, MdKAI2 positively regulates fatty acids, esters, and terpenoids and negatively regulates metabolites of amino acids, amides and alcohols, and the MAPK signaling pathway may mediate this process. The study has provided a new direction for the industrial application of KAR1 in apples and resistance breeding based on the gene of MdKAI2. [ABSTRACT FROM AUTHOR]

SUMMARY Tea plants are perennial evergreen woody crops that originated in low latitudes but have spread to high latitudes. Bud dormancy is an important adaptation mechanism to low temperatures, and its timing is economically significant for tea production. However, the core molecular networks regulating dormancy and bud break in tea plants remain unclear. In the present study, a MADS‐box transcription factor CsMADS27 was identified in tea plants. Gene and phenotype characterizations following ectopic overexpression and endogenous silencing experiments are consistent with a role for CsMADS27 in dormancy and sprouting in different tea cultivars. Furthermore, CsDJC23 was found to be a downstream target of CsMADS27 and implicated in bud sprouting. Based on yeast one‐hybrid screening and comprehensive verification, CsCBF1 and CsZHD9 were identified as upstream transcriptional inhibitors and activators of CsMADS27, respectively, with the two proteins showing direct interactions and competitive binding effects. Histone acetylation (H3K27Ac) in the first exon and intron regions of CsMADS27 was associated with a positive role in CsMADS27 expression. These results revealed that CsMADS27 is a key transcription factor involved in the regulation of dormancy and bud break. Furthermore, the CsCBF1/CsZHD9‐CsMADS27 module plays a critical role in sensing environmental factors and accurately regulating the growth and development of overwintering buds in tea plants. [ABSTRACT FROM AUTHOR]

In this study, we used high-throughput sequencing technology to analyze the diversity and predict gene function of intestinal bacteria from captive black-capped capuchin in two wildlife parks. The results showed that the composition of intestinal gut bacterial diversity was significantly higher in black-capped capuchins monkeys from ES than HT. Moreover, high similarity of intestinal bacteria of black-capped capuchins at the same site was found by sample stratum clustering. Black-capped capuchins intestinal bacteria can be clustered into 2296 ASVs, belonging to 16 phyla, 99 families and 210 genera, respectively. The dominant phyla were Bacteroidota and Firmicutes in the intestinal bacteria of black-capped capuchins, but there were differences in the dominant bacterial families and dominant bacterial genera between ES and HT, and the bacterial families with significant differences had correspondence with bacterial genera. The results provide a basis for the study of intestinal bacteria in black-capped capuchins and the isolation and purification of dominant bacteria. The results of this study fill the gap in the study of intestinal bacterial diversity and its gene functions in black-capped capuchins, and provide a reference for the isolation of dominant bacteria in black-capped capuchins' intestinal microbes and the discovery and study of novel functional genes. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an essential plant phytohormone for plant growth and development. It is involved in important processes such as the regulation of seed germination and dormancy, root growth, stomatal closure, leaf senescence, and abscission. Its function is mainly achieved through a series of signal transductions, and the first step of its signal transduction depends on the binding of its receptor. PYR1-like protein 9 (PYL9) serves as a key receptor in the ABA signaling pathway, positioned upstream. Upon the detection of ABA signals, it suppresses the activity of protein phosphatase 2C (PP2C), thereby activating SNF2-related protein kinase (SnRK2) and facilitating downstream gene's expression to initiate the ABA signaling cascade. This pivotal role contributes significantly to the regulation of plant stress resistance, as well as growth and development, by modulating abscisic acid signal transduction, cellular metabolism, and physiological and biochemical responses within plants. This paper provides a comprehensive review of the regulatory role of the ABA in plant growth, interactions with different hormone signals, synthesis and metabolic pathways, signal transduction composition and function, as well as the structure, functional characteristics, and important role and existing issues of ABA receptor PYL9 in stress response. The paper also prospects its application in future. It is also anticipated to offer a theoretical basis for systematic research on the function of ABA receptor gene PYL9 and mechanisms of plant antiviral defense reactions. [ABSTRACT FROM AUTHOR]

UDP-glucose pyrophosphorylase is ubiquitously present in animals, plants and bacteria and plays essential role in biosynthesis of UDP-glucose which is a fundamentally important molecule in biology. In this paper, we cloned an E. ulmoides UGP1 gene and alignment of the EuUGP1 protein showed it belonged to A-type UGPase (UGPase-A) with characteristics of cytoplasm-localization. Further study showed the EuUGP1 introduction improved UGPase activity and UDP-Glc content in transgenic tobaccos, which subsequently acted as the donor for synthesis of glucose, sucrose, and cellulose, etc. Actually, the EuUGP1-overexpression increased the contents of glucose, cellulose, sucrose and soluble sugar in transgenic tobaccos. Transcriptome data showed that the differentially expressed genes (DEGs) were mainly enriched in metabolic pathway and secondary metabolic pathway, especially the genes related to glycosyltransferase activity (e.g. UGT) being significantly up-regulated. Further, metabolomic data showed that the differentially accumulated metabolites (DAMs) were also mainly enriched in metabolic pathway, secondary metabolic pathway and flavonoid metabolic pathway, and the content of secondary metabolites in transgenic tobaccos was significantly higher than that in wild-type plants. Among these metabolites, the flavonoids and their derivatives including glycosides metabolites accounted for 34% compared with other secondary metabolites, which suggesting EuUGP1 could significantly regulate the UDP-Glc accumulation and the latter provided free glucoside for flavonoid glycosides synthesis. These findings provide a theoretical basis for further study of the function of E. ulmoides EuUGP1 gene.Key message: The overexpression of EuUGP1 augmented the activity of UGPase and the level of UDP-Glc, concurrently elevating the levels of sucrose, glucose, and soluble sugars. At the same time, the content of flavonoids was increased. [ABSTRACT FROM AUTHOR]

B-box transcription factors (TFs) in plants are essential for circadian rhythm regulation, abiotic stress responses, hormonal signaling pathways, secondary metabolism, photomorphogenesis, and anthocyanin formation. Here, by blasting the AtBBX21 gene sequence, we identified a total of 18 BBX21 genes from five distinct Brassica species (Arabidopsis thaliana, Brassica rapa, Brassica oleracea, Brassica napus, and Brassica juncea). The BrBBX21-1 gene is most closely linked to the AtBBX21 gene based on phylogeny and protein sequence similarities. The BrBBX21-1 gene, which encodes a polypeptide of 319 amino acids, was identified from Zicaitai (Brassica rapa ssp. purpuraria) and functionally characterized. BrBBX21-1 was localized within the nucleus, and its overexpression in Arabidopsis augmented anthocyanin accumulation in both leaves and seeds. We further performed an RNA-seq analysis between the BrBBX21-OE and WT A. thaliana to identify the key regulators involved in anthocyanin accumulation. In detail, a total of 7583 genes demonstrated differential expression, comprising 4351 that were upregulated and 3232 that were downregulated. Out of 7583 DEGs, 81 F-box protein genes and 9 B-box protein genes were either up- or downregulated. Additionally, 7583 differentially expressed genes (DEGs) were associated with 109 KEGG pathways, notably including plant hormone signal transduction, the biosynthesis of secondary metabolites, metabolic pathways, glutathione metabolism, and starch and sucrose metabolism, which were considerably enriched. A transcriptome analysis led us to identify several structural genes, including DFRA, GSTF12, UGT75C1, FLS1, CHI1, 4CL3, and PAL1, and transcription factors, MYB90, TT8, and HY5, that are regulated by the overexpression of the BrBBX21-1 gene and involved in anthocyanin biosynthesis. Altogether, these findings demonstrate the beneficial regulatory function of BrBBX21-1 in anthocyanin accumulation and offer valuable information about the basis for breeding superior Brassica crops. [ABSTRACT FROM AUTHOR]

Superoxide dismutase (SOD), a critical enzyme within the plant antioxidant defense system, serves as the primary shield against reactive oxygen species (ROS) under adverse environmental conditions. However, the characterization of GbSODs in ginkgo remains incomplete. In our study, a total of eight GbSODs were identified, which were unevenly distributed across 10 chromosomes and predominantly localized on the plasma membrane. Our phylogenetic analysis revealed that GbSODs from ginkgo and other species form three distinct groups, each characterized by specific binding domains and supported by relatively high bootstrap values. Our promoter analysis indicated that all GbSODs contained multiple cis-elements related to various abiotic stress responses. Our systemic analysis revealed that only one pair of GbSODs underwent segmental duplications. The transcriptome analysis showed the variable expression of GbSODs across different tissues, which were significantly influenced by drought, ultraviolet (UV), and salt stress, with GbSOD8 showing a particularly notable induction. Transgenic plants that overexpressed GbSOD8 exhibited enhanced antioxidant enzyme activities, elevated proline levels, and reduced malondialdehyde (MDA) content, collectively contributing to their improved salt tolerance compared to wild-type plants. Our findings expand the comprehensive understanding of the GbSOD gene family and provide a solid foundation for further elucidating the molecular mechanisms of GbSODs under salt stress. [ABSTRACT FROM AUTHOR]

Auxin response factors (ARFs) are important transcription factors that regulate the expression of auxin response genes, thus play crucial roles in plant growth and development. However, the functions of ARF genes in bermudagrass (Cynodon dactylon L.), a turfgrass species of great economic value, remain poorly understood. In this study, a total of 86 CdARF genes were identified from the C. dactylon genome and were categorized into five groups according to their phylogenetic relationships. The five groups of CdARF genes exhibited specific gene structure and protein domain characteristics, and showed distinct gene expression patterns in different organs, wild accessions and under different stress treatments. Among the 86 CdARF genes, the CdARF6-B2 gene encoded an N-terminally truncated group V ARF protein with high sequence similarity to AtARF2 and OsARF24. The CdARF6-B2 gene was highly expressed in the aboveground vegetative organs (leaf, shoot and stolon) and weakly expressed in the root. The CdARF6-B2 protein was localized in the nucleus but showed no transactivation activity, although its middle region had a strong transactivation activity. Ectopic expression of CdARF6-B2 inhibited the vegetative growth of transgenic Arabidopsis plants possibly through down-regulating the expression of auxin transport-related PIN3 gene and impeding the polar transport of auxin. These results not only established solid foundations to characterize the regulatory mechanism of auxin signaling in the growth and development of bermudagrass but also provided new insights into the function of ARF genes in plants. [ABSTRACT FROM AUTHOR]

Abstract Background Leucine-rich repeat (LRR) extensins (LRXs), which are cell wall-localized chimeric extensin proteins, are essential for the development of plants and their resistance to stress. Despite the significance of these genes, an extensive genome-wide analysis of the LRX gene family in grapevine (Vitis vinifera L.) is lacking. Results We here detected 14 grapevine LRX genes and classified them into four groups through phylogenetic analysis. Then, their physiological and biochemical properties and gene/protein structures were analyzed. According to synteny analysis, tandem and segmental duplications have appreciably affected the expansion of the grapevine LRX gene family. On investigating tissue-specific expression profiles and cis-regulatory elements, we observed that VvLRXs likely serve as regulators of both the growth of grapevines and their responses to various environmental stresses. Salt stress treatments induced the expression of several VvLRXs, and VvLRX7 expression was the most significantly upregulated. Furthermore, VvLRX7 expression was positively correlated with the salt tolerance of grape rootstocks. VvLRX7 overexpression in Arabidopsis markedly enhanced its salt tolerance. Conclusion This study provides a general understanding of the characteristics and evolution of the LRX gene family in grapevine. VvLRX7 may function as a positive regulator of plant’s response to salt stress. These findings offer a basis for future studies on the function of grapevine LRXs and their role in improving salt stress tolerance in grapevine.

[Objective] Tuber mustard has high economic value. Investigating the function of the jasmonic acid signaling pathway gene JAZ8 in response to salt stress can provide genetic resources for breeding saltresistant varieties of tuber mustard. [Methods] The gene structure, promoter cis-acting elements, and the physical and chemical properties of JAZ8 were analyzed by bioinformatics methods. JAZ8 gene was cloned and the overexpression vector was constructed. The JAZ8 gene of tuber mustard was overexpressed in the model plant Arabidopsis thaliana and the function of JAZ8 gene in Arabidopsis in response to salt stress was analyzed. [Results] The JAZ8 gene coding sequence was 396 bp, encoding 131 amino acids and containing TIFY and CCT-2 domains. The gene promoter contained cis-acting elements that respond to abscisic acid, salicylic acid, and participate in defense and stress response. Phenotypic analysis of the transgenic plants showed that Arabidopsis plants overexpressing BjuJAZ8 showed a more sensitive phenotype to salt stress during germination and greening. [Conclusion] Overexpression of JAZ8 from tuber mustard enhanced the sensitivity of Arabidopsis to salt stress.

Abstract Background In research to improve the quality of transgenic crops, it is often necessary to introduce multiple functionally related genes into recipient plants simultaneously to improve crop genetic traits effectively. Compared with unidirectional promoters, bidirectional promoters simultaneously regulate the expression of multiple genes and improve the efficiency of biotechnology. Therefore, in this study, bidirectional gene pairs were systematically analyzed in Gossypium hirsutum TM-1, and the structure, function and evolutionary relationships of the bidirectional genes were analyzed. The endogenous bidirectional promoters of cotton were mined, and their specific regulatory elements and biological functions were explored to provide useful promoter resources and a theoretical basis for cultivating new cotton germplasms with excellent fiber quality. Results Using an improved search model, a total of 1,383 bidirectional transcript pairs were identified in the Gossypium hirsutum TM-1 genome, and their gene structure and functional annotations were systematically analyzed. Thirty bidirectional intergenic sequences were randomly screened for promoter activity analysis via a transient expression system, and 25 intergenic sequences were found to have bidirectional promoter activity. Comparative analysis of the bidirectional gene profiles of the four cotton subspecies revealed that these subspecies presented abundant bidirectional gene pairs with high homology and that the bidirectional genes in the cotton subspecies were more similar in terms of their molecular functions, cellular components and biological processes. In addition, parallel analysis of bidirectional genes in dicotyledons and monocotyledons revealed that abundant bidirectional gene pairs exist in different species. Although the total number of orthologous bidirectional genes was similar, there was a significant difference in the number of orthologous bidirectional gene pairs between dicotyledons and monocotyledons. This evolutionary analysis of the function and structure of homologous bidirectional gene pairs in different varieties and different subspecies of the same species revealed potential pathways by which these gene pairs originated, which may be necessary for the evolution of a new species. Conclusion In this study, many bidirectional gene pairs in Gossypium hirsutum TM-1 were identified using computer programming, and systematic analysis was conducted to explore their functions and evolutionary relationships. In addition, the promoter activity of the bidirectional intergenic sequences was verified. The combination of computer programming screening, experimental validation and other methods is expected to provide preferred bidirectional promoters for transgenic breeding work via multigene cotransformation methods, and this information is valuable for genetic engineering research and applications.

Background: Leucine-rich repeat (LRR) extensins (LRXs), which are cell wall-localized chimeric extensin proteins, are essential for the development of plants and their resistance to stress. Despite the significance of these genes, an extensive genome-wide analysis of the LRX gene family in grapevine (Vitis vinifera L.) is lacking. Results: We here detected 14 grapevine LRX genes and classified them into four groups through phylogenetic analysis. Then, their physiological and biochemical properties and gene/protein structures were analyzed. According to synteny analysis, tandem and segmental duplications have appreciably affected the expansion of the grapevine LRX gene family. On investigating tissue-specific expression profiles and cis-regulatory elements, we observed that VvLRXs likely serve as regulators of both the growth of grapevines and their responses to various environmental stresses. Salt stress treatments induced the expression of several VvLRXs, and VvLRX7 expression was the most significantly upregulated. Furthermore, VvLRX7 expression was positively correlated with the salt tolerance of grape rootstocks. VvLRX7 overexpression in Arabidopsis markedly enhanced its salt tolerance. Conclusion: This study provides a general understanding of the characteristics and evolution of the LRX gene family in grapevine. VvLRX7 may function as a positive regulator of plant's response to salt stress. These findings offer a basis for future studies on the function of grapevine LRXs and their role in improving salt stress tolerance in grapevine. [ABSTRACT FROM AUTHOR]

Simple Summary: The brown planthopper (Nilaparvata lugens) is a major pest threatening rice crops, especially in Asia and Africa, causing damage and transmitting harmful viruses. Current control strategies, such as the use of chemical pesticides, are becoming increasingly ineffective due to the development of resistance in pest populations. RNA interference (RNAi) is a promising alternative for silencing essential genes in pests. This study focuses on the NlCPSF30 gene (homolog of mammalian CPSF30), which is crucial for mRNA processing in brown planthoppers. By using RNAi to knock down the NlCPSF30 gene, we observed marked reductions in survival rates and notable developmental defects in the pests. These findings highlight the potential of targeting NlCPSF30 for developing RNAi-based pest control strategies. The brown planthopper (Nilaparvata lugens) is a major pest threatening global rice production, significantly reducing yields annually. As N. lugens increasingly develops resistance to conventional control methods, such as chemical pesticides, there is an urgent need for innovative and sustainable pest management strategies. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a key protein involved in mRNA 3′ end processing, yet its function in N. lugens remains poorly understood. This study aims to elucidate the role of CPSF30 in the growth and development of N. lugens and evaluate its potential as a target for RNA interference (RNAi)-based pest control strategies. We cloned and characterized the cDNA sequence of NlCPSF30, which encodes a protein of 341 amino acids containing five CCCH zinc-finger domains and two CCHC zinc-knuckle domains. Sequence alignment revealed that NlCPSF30 is highly conserved among insect species, particularly in the zinc-finger domains essential for RNA binding and processing. Phylogenetic analysis showed that NlCPSF30 is closely related to CPSF30 proteins from other hemipteran species. Expression analysis indicated that NlCPSF30 is most highly expressed in the fat body and during the adult stage, with significantly higher expression in females than in males. RNAi-mediated silencing of NlCPSF30 in third-instar nymphs resulted in severe phenotypic abnormalities, including disrupted molting and increased mortality following injection of double-stranded RNA (dsRNA) targeting NlCPSF30. Moreover, it influenced the expression of genes associated with hormone regulation, namely NlHry, NlE93, and NlKr-h1. These results suggest that NlCPSF30 is integral to critical physiological processes, with its disruption leading to increased mortality. Our findings identify NlCPSF30 as an essential gene for N. lugens' survival and a promising target for RNAi-based pest management strategies. This study provides a valuable molecular target and theoretical insights for developing RNAi-based control methods against N. lugens. [ABSTRACT FROM AUTHOR]

The plant MADS-box transcription factors constitute a class of proteins that possess the MADS-box structural domain. These proteins play crucial roles in regulating plant growth and development, as well as in responding to adversity. The biological functions of rice MADS-box transcription factors have gained significant attention, leading to numerous research advancements. The functions of at least 33 of the 75 known rice MADS-box family members have been studied and reported, and they play pivotal roles in flower development, spikelet development, plant height morphogenesis, root development, flowering habit, seed development, and responses to both biotic and abiotic stresses,such as drought, salinity, high temperature, low temperature, and rice blast. Research has shown that MADS-box members form a complex regulatory network by interacting with each other or with other transcription factors, which mainly involves phytohormone signaling, reactive oxygen species homeostasis, osmotic adjustment, ubiquitination, DNA methylation and the synergistic effects among them. It was found that most MADS-box family members are involved in the regulation of growth and development, especially flower development, and there is redundancy in the biological functions of some of them, but they may play roles in different developmental stages, tissue types, or environmental conditions, and more in-depth analysis of the functional specificity and redundancy of these genes is needed in the future in order to reveal their unique roles in the growth and development of rice. However, there are still numerous members of the rice MADS-box family whose functions are unknown, and more research is needed to reveal their roles. Notably, some rice MADS-box members show potential application value, and the efficient utilization of these genetic resources to improve traits such as yield, quality, and stress tolerance of rice through prime editing and heavy ion beam irradiation mutagenesis technology is an important direction for future research. In this paper, we summarize the biological functions of rice MADS-box transcription factors, aiming to provide insights and strategies for rice molecular breeding. [ABSTRACT FROM AUTHOR]

Due to the growing focus on daily hygiene practices, sodium dodecyl sulfate (SDS), a widely used surfactant, is increasingly found in domestic sewage and rainfall runoff. Upon entering the lake–terrestrial ecotone, SDS affects the composition, abundance, and functional capacity of soil bacterial communities due to its bacteriostatic properties. To investigate the effects of long-term discharge of sewage containing low concentrations of SDS on microorganisms in the lake–terrestrial ecotone, alterations in bacterial community structure, functional genes, and biomass were examined using a simulated continuous pollutant input. The results indicated the following: (1) The degradation rate of sodium dodecyl sulfate (SDS) by soil microorganisms in the lake–terrestrial ecotone under long-term and low concentrations of SDS stress ranged from 11 to 16 mg/kg·d. (2) The effects of low concentrations and long-term SDS stress on bacterial community structure and gene function in the lake–terrestrial ecotone differed significantly from those of short-term pollution. The damage to microbial-promoted material cycling in the lake–terrestrial ecotone was more severe; however, the proliferation of pathogenic bacteria remained continuously suppressed. (3) Soil bacteria in the lake–terrestrial ecotone responded to the stress of long-term and low concentrations of SDS primarily by enhancing chemotaxis and tolerance. [ABSTRACT FROM AUTHOR]

Cinnamyl alcohol dehydrogenase (CAD) is a crucial enzyme in the final stage of lignin monomer biosynthesis. This study focuses on the CAD gene family within Gossypium hirsutum. Through comprehensive genomic analysis, we identified 29 GhiCAD genes within the Gossypium hirsutum genome using a bioinformatics approach. Phylogenetic analysis revealed that the GhiCAD family can be categorized into four subgroups, which are closest to the evolutionary relationship with Arabidopsis thaliana. There are multiple cis-acting elements on the promoters of GhiCAD genes associated with abiotic stress responses. Some GhiCAD genes demonstrated high expression in various tissues like root, leaf, and sepal, as well as in fiber and ovule at different developmental stages (10 days post anthesis (DPA), 15 DPA, 20 DPA, 25 DPA). The transcript levels of GhiCAD23 were notably elevated when exposed to PEG treatment and drought stress (DS). GhiCAD23 is also co-expressed with many known drought response genes, suggesting its involvement in the plant's reaction to DS. Employing virus-induced gene silencing (VIGS) technology to silence the GhiCAD23 gene, it was found that silencing GhiCAD23 reduced the tolerance of cotton to DS. Under DS, the relative leaf water content, superoxide dismutase (SOD), and catalase (CAT) enzyme activities of the GhiCAD23-silenced cotton plants were decreased by 31.84%, 30.22% and 14.19%, respectively, while malondialdehyde (MDA) was increased by 72.16% compared with the control cohort. Drought promotes the accumulation of lignin, and it was found that silencing the GhiCAD23 reduces lignin accumulation in cotton under DS. The analysis of phenotypic and physiological indicators indicates that GhiCAD23 is vital in cotton's resistance to DS. This investigation provides an important reference for future comprehensive exploration of the GhiCAD23 gene's function in cotton's DS response mechanism. [ABSTRACT FROM AUTHOR]

Elevated salinity negatively impacts plant growth and yield, presenting substantial challenges to agricultural and forestry productivity. The bHLH transcription factor family is vital for plants to cope with various abiotic stresses. However, it remains uncertain whether bHLH transcription factors can regulate salt stress in Populus ussuriensis. In the following study, a salt-induced bHLH transcription factor PubHLH66 was identified from P. ussuriensis. PubHLH66 has a typical and conserved bHLH domain. Subcellular localization and yeast two-hybrid (Y2H) assays confirmed that it is a nucleus-localized transactivator and the activation region is located at the N-terminus. PubHLH66-OE and PubHLH66-SRDX transgenic P. ussuriensis were obtained through Agrobacterium-mediated leaf disc transformation. Morphological and physiological results demonstrated that PubHLH66-OE enhanced salinity tolerance, as indicated by reduced electrolyte leakage (EL), malondialdehyde (MDA), and H2O2 levels, along with increased proline contents and activities of peroxidase (POD) and superoxide dismutase (SOD). In contrast, PuHLH66-SRDX poplar showed decreased salt tolerance. Quantitative real-time PCR (RT-qPCR) confirmed that PubHLH66 enhanced salt tolerance by regulating the expression of genes such as PuSOD, PuPOD, and PuP5CS, resulting in reduced reactive oxygen species (ROS) accumulation and an improved osmotic potential. Thus, PubHLH66 could be a candidate gene for molecular breeding to enhance salt tolerance in plants. These results laid a foundation for exploring the mechanisms of salt tolerance in P. ussuriensis, facilitating the development of more salt-tolerant trees to combat the increasing issue of soil salinization globally. [ABSTRACT FROM AUTHOR]

Aphis gossypii is one of the most economically important agricultural pests that cause serious crop losses worldwide, and the indiscriminate chemical application causes resistance development in A. gossypii, a major obstacle to successful control. In this study, we selected the up-regulated expression gene AgJHAMT , which was enriched into juvenile hormone pathway though transcriptome sequencing analysis of the cotton aphids that fed on transgenic cotton lines expressing ds AgCYP6CY3 (the TG cotton). The AgJHAMT gene was overexpressed in cotton aphids which fed on the TG cotton, and its expression profile during the nymphs was clarified. Then, silencing AgJHAMT could advance the developmental period of cotton aphids by 0.5 days compared with control groups. The T and t values of cotton aphids in the dsJHAMT treatment group (6.88 ± 0.15, 1.65 ± 0.06) were significantly shorter than that of the sprayed H2O control group (7.6 ± 0.14, 1.97 ± 0.09) (P < 0.05), respectively. The fast growth caused by AgJHAMT silencing was rescued by applying the JH analogue, methoprene. Overall, these findings clarified the function of AgJHAMT in the developmental period of A. gossypii. This study contributes to further clarify the molecular mechanisms of delaying the growth and development of cotton aphids by the transgenic cotton lines expressing ds AgCYP6CY3. [ABSTRACT FROM AUTHOR]

Cucumber (Cucumis sativus L.) is a major vegetable crop grown globally, with a cultivation history of more than 3000 years. The limited genetic diversity, low rate of intraspecific variation, and extended periods of traditional breeding have resulted in slow progress in their genetic research and the development of new varieties. Gamma (γ)-ray irradiation potentially accelerates the breeding progress; however, the biological and molecular effects of γ-ray irradiation on cucumbers are unknown. Exposing cucumber seeds to 0, 50, 100, 150, 200, and 250 Gy doses of 60Co-γ-ray irradiation, this study aimed to investigate the resulting phenotype and physiological characteristics of seedling treatment to determine the optimal irradiation dose. The results showed that low irradiation doses (50–100 Gy) enhanced root growth, hypocotyl elongation, and lateral root numbers, promoting seedling growth. However, high irradiation doses (150–250 Gy) significantly inhibited seed germination and growth, decreasing the survival rate of seedlings. More than 100 Gy irradiation significantly decreased the total chlorophyll content while increasing the malondialdehyde (MDA) and H2O2 content in cucumber. Transcriptome sequencing analysis at 0, 50, 100, 150, 200, and 250 Gy doses showed that gene expression significantly differed between low and high irradiation doses. Gene Ontology enrichment and functional pathway enrichment analyses revealed that the auxin response pathway played a crucial role in seedling growth under low irradiation doses. Further, gene function analysis revealed that small auxin up-regulated gene CsSAUR37 was a key gene that was overexpressed in response to low irradiation doses, promoting primary root elongation and enhancing lateral root numbers by regulating the expression of protein phosphatase 2Cs (PP2Cs) and auxin synthesis genes.Key message: Low doses of γ-ray irradiation promoted seedling growth; CsSAUR37 was identified and overexpression of CsSAUR37 was found to promote primary root elongation and hypocotyl elongation, and increases in lateral root numbers; Cucumber exhibited phenotypic variations under γ-ray irradiation. These findings provided a foundation for constructing cucumber mutant libraries and excellent materials for understanding the mechanisms underlying γ-ray irradiation. [ABSTRACT FROM AUTHOR]

Melon (Cucumis melo) includes more than ten botanical groups, many of which feature netting ornamentation on the surface of mature fruit. Ripe melons display a netted skin that signifies their ripeness and readiness for consumption. Previously, we identified SKIN NETTING (CmSN), which encodes an EamA-like transporter family protein, as the candidate gene controlling fruit skin netting formation in melon, while its biological functions remain unclear. In this study, we demonstrated that the expression of the CmSN gene was considerably lower in netted melons compared to smooth-skinned melons, indicating a negative correlation between CmSN expression and netting formation. Subsequently, we employed transient overexpression and virus-induced gene silencing (VIGS) experiments to explore the role of CmSN gene during fruit development. Overexpression of the CmSN gene inhibited netting development, whereas silencing it promoted netting formation. Using heterologous transformation in tomato, we further confirmed the effect of the CmSN gene on rind texture and toughness, as these tomatoes exhibited rougher and tougher skins. Analysis with near-isogenic lines (NILs) revealed that CmSN gene-bearing fruits (NIL_CmSN) possessed significantly harder rinds than the control smooth-skinned variety HB42, underscoring the role of CmSN in enhancing rind protection. Together, our research offers essential insights into the netting formation and genetic improvement of melon fruits. [ABSTRACT FROM AUTHOR]

To meet the demands of a rising human population, plant breeders will need to develop improved crop varieties that maximize yield in the face of increasing pressure on crop production. Historically, the optimization of crop root architecture has represented a challenging breeding target due to the inaccessibility of the root systems. Root hairs, single cell projections from the root epidermis, are perhaps the most overlooked component of root architecture traits. Root hairs play a central role in facilitating water, nutrient uptake, and soil cohesion. Current root hair architectures may be suboptimal under future agricultural production regimes, coupled with an increasingly variable climate. Here, we review the genetic control of root hair development in the world's three most important crops—rice, maize, and wheat—and highlight conservation of gene function between monocots and the model dicot species Arabidopsis. Advances in genomic techniques including gene editing combined with traditional plant breeding methods have the potential to overcome many inherent issues associated with the design of improved root hair architectures. Ultimately, this will enable detailed characterization of the effects of contrasting root hair morphology strategies on crop yield and resilience, and the development of new varieties better adapted to deliver future food security. [ABSTRACT FROM AUTHOR]

Dryopteris fragrans (L.) Schott has anti-inflammatory and antioxidant properties, and terpenoids are important components of its active constituents. The methyl-D-erythritol 4-phosphate (MEP) pathway is one of the major pathways for the synthesis of terpene precursors in plants, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in this pathway. DXS has been shown to be associated with increased stress tolerance in plants. In this experiment, two DXS genes were extracted from the D. fragrans transcriptome and named DfDXS1 and DfDXS2. Based on phylogenetic tree and conserved motif analyses, DXS was shown to be highly conserved evolutionarily and its localization to chloroplasts was determined by subcellular localization. Prokaryotic expression results showed that the number and growth status of recombinant colonies were better than the control under 400 mM NaCl salt stress and 800 mM mannitol-simulated drought stress. In addition, the DfDXS1 and DfDXS2 transgenic tobacco plants showed improved resistance to drought and salt stress. DfDXS1 and DfDXS2 responded strongly to methyl jasmonate (MeJA) and PEG-mimicked drought stress following exogenous hormone and abiotic stress treatments of D. fragrans. The transcriptional active sites were investigated by dual luciferase and GUS staining assays, and the results showed that the STRE element (AGGGG), the ABRE element (ACGTGGC), and the MYC element (CATTTG) were the important transcriptional active sites in the promoters of the two DXS genes, which were closely associated with hormone response and abiotic stress. These results suggest that the DfDXS gene of D. fragrans plays an important role in hormone signaling and response to stress. This study provides a reference for analyzing the molecular mechanisms of stress tolerance in D. fragrans. [ABSTRACT FROM AUTHOR]

Background: The variant call format (VCF) file is a structured and comprehensive text file crucial for researchers and clinicians in interpreting and understanding genomic variation data. It contains essential information about variant positions in the genome, along with alleles, genotype calls, and quality scores. Analyzing and visualizing these files, however, poses significant challenges due to the need for diverse resources and robust features for in-depth exploration. Results: To address these challenges, we introduce variant graph craft (VGC), a VCF file visualization and analysis tool. VGC offers a wide range of features for exploring genetic variations, including extraction of variant data, intuitive visualization, and graphical representation of samples with genotype information. VGC is designed primarily for the analysis of patient cohorts, but it can also be adapted for use with individual probands or families. It integrates seamlessly with external resources, providing insights into gene function and variant frequencies in sample data. VGC includes gene function and pathway information from Molecular Signatures Database (MSigDB) for GO terms, KEGG, Biocarta, Pathway Interaction Database, and Reactome. Additionally, it dynamically links to gnomAD for variant information and incorporates ClinVar data for pathogenic variant information. VGC supports the Human Genome Assembly Hg37 and Hg38, ensuring compatibility with a wide range of data sets, and accommodates various approaches to exploring genetic variation data. It can be tailored to specific user needs with optional phenotype input data. Conclusions: In summary, VGC provides a comprehensive set of features tailored to researchers working with genomic variation data. Its intuitive interface, rapid filtering capabilities, and the flexibility to perform queries using custom groups make it an effective tool in identifying variants potentially associated with diseases. VGC operates locally, ensuring data security and privacy by eliminating the need for cloud-based VCF uploads, making it a secure and user-friendly tool. It is freely available at https://github.com/alperuzun/VGC. [ABSTRACT FROM AUTHOR]

The fall armyworm (Spodoptera frugiperda) poses a substantial threat to many important crops worldwide, emphasizing the need to develop and implement advanced technologies for effective pest control. CRISPR/Cas9, derived from the bacterial adaptive immune system, is a prominent tool used for genome editing in living organisms. Due to its high specificity and adaptability, the CRISPR/Cas9 system has been used in various functional gene studies through gene knockout and applied in research to engineer phenotypes that may cause economical losses. The practical application of CRISPR/Cas9 in diverse insect orders has also provided opportunities for developing strategies for genetic pest control, such as gene drive and the precision-guided sterile insect technique (pgSIT). In this review, a comprehensive overview of the recent progress in the application of the CRISPR/Cas9 system for functional gene studies in S. frugiperda is presented. We outline the fundamental principles of applying CRISPR/Cas9 in S. frugiperda through embryonic microinjection and highlight the application of CRISPR/Cas9 in the study of genes associated with diverse biological aspects, including body color, insecticide resistance, olfactory behavior, sex determination, development, and RNAi. The ability of CRISPR/Cas9 technology to induce sterility, disrupt developmental stages, and influence mating behaviors illustrates its comprehensive roles in pest management strategies. Furthermore, this review addresses the limitations of the CRISPR/Cas9 system in studying gene function in S. frugiperda and explores its future potential as a promising tool for controlling this insect pest. [ABSTRACT FROM AUTHOR]

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types—susceptible, immune, and "Schrödinger" (conditional)—based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development. [ABSTRACT FROM AUTHOR]

Systematic characterization of biological effects to genetic perturbation is essential to the application of molecular biology and biomedicine. However, the experimental exhaustion of genetic perturbations on the genome-wide scale is challenging. Here, we show TranscriptionNet, a deep learning model that integrates multiple biological networks to systematically predict transcriptional profiles to three types of genetic perturbations based on transcriptional profiles induced by genetic perturbations in the L1000 project: RNA interference, clustered regularly interspaced short palindromic repeat, and overexpression. TranscriptionNet performs better than existing approaches in predicting inducible gene expression changes for all three types of genetic perturbations. TranscriptionNet can predict transcriptional profiles for all genes in existing biological networks and increases perturbational gene expression changes for each type of genetic perturbation from a few thousand to 26 945 genes. TranscriptionNet demonstrates strong generalization ability when comparing predicted and true gene expression changes on different external tasks. Overall, TranscriptionNet can systemically predict transcriptional consequences induced by perturbing genes on a genome-wide scale and thus holds promise to systemically detect gene function and enhance drug development and target discovery. [ABSTRACT FROM AUTHOR]

Background: In research to improve the quality of transgenic crops, it is often necessary to introduce multiple functionally related genes into recipient plants simultaneously to improve crop genetic traits effectively. Compared with unidirectional promoters, bidirectional promoters simultaneously regulate the expression of multiple genes and improve the efficiency of biotechnology. Therefore, in this study, bidirectional gene pairs were systematically analyzed in Gossypium hirsutum TM-1, and the structure, function and evolutionary relationships of the bidirectional genes were analyzed. The endogenous bidirectional promoters of cotton were mined, and their specific regulatory elements and biological functions were explored to provide useful promoter resources and a theoretical basis for cultivating new cotton germplasms with excellent fiber quality. Results: Using an improved search model, a total of 1,383 bidirectional transcript pairs were identified in the Gossypium hirsutum TM-1 genome, and their gene structure and functional annotations were systematically analyzed. Thirty bidirectional intergenic sequences were randomly screened for promoter activity analysis via a transient expression system, and 25 intergenic sequences were found to have bidirectional promoter activity. Comparative analysis of the bidirectional gene profiles of the four cotton subspecies revealed that these subspecies presented abundant bidirectional gene pairs with high homology and that the bidirectional genes in the cotton subspecies were more similar in terms of their molecular functions, cellular components and biological processes. In addition, parallel analysis of bidirectional genes in dicotyledons and monocotyledons revealed that abundant bidirectional gene pairs exist in different species. Although the total number of orthologous bidirectional genes was similar, there was a significant difference in the number of orthologous bidirectional gene pairs between dicotyledons and monocotyledons. This evolutionary analysis of the function and structure of homologous bidirectional gene pairs in different varieties and different subspecies of the same species revealed potential pathways by which these gene pairs originated, which may be necessary for the evolution of a new species. Conclusion: In this study, many bidirectional gene pairs in Gossypium hirsutum TM-1 were identified using computer programming, and systematic analysis was conducted to explore their functions and evolutionary relationships. In addition, the promoter activity of the bidirectional intergenic sequences was verified. The combination of computer programming screening, experimental validation and other methods is expected to provide preferred bidirectional promoters for transgenic breeding work via multigene cotransformation methods, and this information is valuable for genetic engineering research and applications. [ABSTRACT FROM AUTHOR]

Alfin-like proteins (ALs) form a plant-specific transcription factor (TF) gene family involved in the regulation of plant growth and development, and abiotic stress response. In this study, 30 ALs were identified in Brassica napus ecotype 'Zhongshuang 11' genome (BnaALs), and unevenly distributed on 15 chromosomes. Structural characteristic analysis showed that all of the BnaALs contained two highly conserved domains: the N terminal DUF3594 domain and the C-terminal PHD-finger domain. The BnaALs were classified into four groups (Group I-IV), supported by conserved intron–exon and protein motif structures in each group. The allopolyploid event between B. oleracea and B. rapa ancestors and the small-scale duplication events in B. napus both contributed to the large BnaALs expansion. The promoter regions of BnaALs contained multiple abiotic stress cis-elements. The BnaALs in I-IV groups were mainly expressed in cotyledon, petal, root, silique, and seed tissues, and the duplicated gene pairs shared highly similar expression patterns. RNA-seq and RT-qPCR analysis showed that BnaALs were obviously induced by low nitrogen (LN) and low phosphorus (LP) treatments in roots. Overexpressing BnaAL02 and BnaAL28 in Arabidopsis demonstrated their functions in response to LN and LP stresses. BnaAL28 enhanced primary roots' (PRs) length and lateral roots' (LRs) number under LP and LN conditions, where BnaAL02 can inhibit LR numbers under the two conditions. They can promote root hair (RH) elongation under LP conditions; however, they suppressed RH elongation under LN conditions. Our result provides new insight into the functional dissection of this family in response to nutrient stresses in plants. [ABSTRACT FROM AUTHOR]

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Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.

IntroductionArthrinium phaeospermum can cause large areas wilted and death of Bambusa pervariabilis × Dendrocalamopsis grandis, resulting in serious ecological and economic losses. Previous studies found that the appressorium of A. phaeospermum must form to invade the host cells and cause disease. A short-chain dehydrogenase/reductase gene has been shown to maintain the osmotic pressure of the appressorium by synthesizing fungal melanin to penetrate the plant epidermis and cause disease. The SDR gene family of A. phaeospermum was found to be highly expressed during the penetration in the transcriptome sequencing results. Still, the relationship with melanin biosynthesis of A. phaeospermum is not clear.MethodsWe aimed to predict the biological function of the SDR gene family in A. phaeospermum, identify key ApSDR genes with pathogenic roles, and explore the pathogenic mechanism. We have characterized the SDR family of A. pheospermum bioinformatically. Candidate ApSDRs screened by transcriptome sequencing were compared by qPCR experiments to obtain key ApSDRs that may play an important role in infestation and adversity resistance. Knockout mutants, the co-knockout mutant, and backfill mutants of key ApSDRs were obtained for phenotypic and stress conditions analysis. We explored and validated the pathogenic mechanisms through cellulose membrane penetration experiments and analysis of melanin-related gene synthesis levels.Results and discussion180 ApSDRs were identified bioinformatically. After screening six candidate ApSDRs with noticeably elevated expression using transcriptome sequencing, qPCR experiments revealed that ApSDR53C2 and ApSDR548U2 had the highest expression. The results of phenotypic and stress conditions analysis indicate that ApSDRs are critical for the growth, development, stress response, and fungicide resistance of A. phaeospermum. The pathogenicity analysis revealed that ApSDR53C2 and ApSDR548U2 play important roles in virulence, with ApSDR53C2 having a stronger effect. A comparison of melanin synthesis levels between wild-type and ΔApSDR53C2 strains showed that ApSDR53C2 positively regulates melanin biosynthesis to promote penetration. The findings demonstrate that ApSDRs are essential for A. phaeospermum to withstand stress and facilitate melanin biosynthesis, which in turn contributes to its virulence.

KAR (Karrikin), a novel plant growth regulator, can be recognized specifically by plants and can activate resistance responses. MdKAI2 is the natural receptor of KARs in apple. Here, we report the identification of osmotic stress resistance in MdKAI2 via the method of genetic transformation. The phenotypic traits, resistance indicators, and transcriptional and metabolic regulation of MdKAI2 were identified. KAR1, a highly active form of KAR, markedly promoted the root growth of Gala cultivar tissue culture‒generated plants, possibly through increases in ABA and TZR contents and decreases in the GA3 content. MdKAI2 was markedly upregulated by PEG stress and significantly promoted the growth of apple calli under nonstress conditions, whereas it was significantly inhibited under 20% PEG stress, as was cell death. MdKAI2 significantly increased the content of total flavonoids, the activity of reactive oxygen species (ROS)‒scavenging enzymes (SOD, POD and CAT), and the content of osmoregulatory substances (soluble protein, soluble sugars and proline). It also inhibited the MDA content and conductivity under osmotic stress. Differentially expressed genes (DEGs), including multiple transcription factors (TFs), such as MYB, bHLH and AP2‒EREBP, are significantly regulated by MdKAI2, and genes involved in the mitogen‒activated protein kinase (MAPK) signaling pathway play crucial roles in the regulation of plant resistance. In addition, pathways such as brassinosteroid (BR) biosynthesis and ABC transporters were downregulated, and the MAPK signaling pathway; plant‒pathogen interaction; cutin, suberin and wax biosynthesis; alpha‒linolenic acid metabolism; and phenylpropanoid biosynthesis were upregulated by MdKAI2. MdKAI2 significantly regulates the levels of lipids, amino acids, terpenoids, benzene, organic acids, carbohydrates, and alkaloids and is involved in the metabolic processes of amino acids, carbohydrates, nucleotides, lipids and secondary metabolites. Furthermore, MdKAI2 positively regulates fatty acids, esters, and terpenoids and negatively regulates metabolites of amino acids, amides and alcohols, and the MAPK signaling pathway may mediate this process. The study has provided a new direction for the industrial application of KAR1 in apples and resistance breeding based on the gene of MdKAI2.

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on host–pathogen interaction.