Evolution of the WRKY Family in Angiosperms and Functional Diversity under Environmental Stress.

The transcription factor is an essential factor for regulating the responses of plants to external stimuli. The WRKY protein is a superfamily of plant transcription factors involved in response to various stresses (e.g., cold, heat, salt, drought, ions, pathogens, and insects). During angiosperm evolution, the number and function of WRKY transcription factors constantly change. After suffering from long-term environmental battering, plants of different evolutionary statuses ultimately retained different numbers of WRKY family members. The WRKY family of proteins is generally divided into three large categories of angiosperms, owing to their conserved domain and three-dimensional structures. The WRKY transcription factors mediate plant adaptation to various environments via participating in various biological pathways, such as ROS (reactive oxygen species) and hormone signaling pathways, further regulating plant enzyme systems, stomatal closure, and leaf shrinkage physiological responses. This article analyzed the evolution of the WRKY family in angiosperms and its functions in responding to various external environments, especially the function and evolution in Magnoliaceae plants. It helps to gain a deeper understanding of the evolution and functional diversity of the WRKY family and provides theoretical and experimental references for studying the molecular mechanisms of environmental stress.

Genome-Wide Analysis of the Liriodendron chinense Hsf Gene Family under Abiotic Stress and Characterization of the LcHsfA2a Gene.

Heat shock factors (Hsfs) play a crucial role in plant defense processes. However, the distribution and functional characteristics of Hsf genes in the relict plant Liriodendron chinense are still unclear. In this study, a total of 19 LcHsfs were identified and divided into three separate subgroups, comprising 10 LcHsfA, 7 LcHsfB, and 2 LcHsfC genes, respectively, based on their phylogenetic tree and the presence/absence of conserved protein domains. Whole-genome duplication and segmental duplication led to an expansion of the LhHsf gene family. The promoters of LcHsf genes are enriched for different types of cis-acting elements, including hormone responsive and abiotic-stress-responsive elements. The expression of LcHsfA3, LcHsfA4b, LcHsfA5, LcHsfB1b, and LcHsfB2b increased significantly as a result of both cold and drought treatments. LcHsfA2a, LcHsfA2b, and LcHsfA7 act as important genes whose expression levels correlate strongly with the expression of the LcHsp70, LcHsp110, and LcAPX genes under heat stress. In addition, we found that transiently transformed 35S:LcHsfA2a seedlings showed significantly lower levels of hydrogen peroxide (H2O2) after heat stress and showed a stronger thermotolerance. This study sheds light on the possible functions of LcHsf genes under abiotic stress and identifies potentially useful genes to target for molecular breeding, in order to develop more stress-resistant varieties.

Genomic survey and expression analysis of LcARFs reveal multiple functions to somatic embryogenesis in Liriodendron.

BACKGROUND: Auxin response factors (ARFs) are critical transcription factors that mediate the auxin signaling pathway and are essential for regulating plant growth. However, there is a lack of understanding regarding the ARF gene family in Liriodendron chinense, a vital species in landscaping and economics. Thus, further research is needed to explore the roles of ARFs in L. chinense and their potential applications in plant development. RESULT: In this study, we have identified 20 LcARF genes that belong to three subfamilies in the genome of L. chinense. The analysis of their conserved domains, gene structure, and phylogeny suggests that LcARFs may be evolutionarily conserved and functionally similar to other plant ARFs. The expression of LcARFs varies in different tissues. Additionally, they are also involved in different developmental stages of somatic embryogenesis. Overexpression of LcARF1, LcARF2a, and LcARF5 led to increased activity within callus. Additionally, our promoter-GFP fusion study indicated that LcARF1 may play a role in embryogenesis. Overall, this study provides insights into the functions of LcARFs in plant development and embryogenesis, which could facilitate the improvement of somatic embryogenesis in L. chinense. CONCLUSION: The research findings presented in this study shed light on the regulatory roles of LcARFs in somatic embryogenesis in L. chinense and may aid in accelerating the breeding process of this tree species. By identifying the specific LcARFs involved in different stages of somatic embryogenesis, this study provides a basis for developing targeted breeding strategies aimed at optimizing somatic embryogenesis in L. chinense, which holds great potential for improving the growth and productivity of this economically important species.

The Identification and Expression Analysis of the Liriodendron chinense F-Box Gene Family.

The F-box gene family is one of the largest gene families in plants, and it plays a crucial role in regulating plant development, reproduction, cellular protein degradation, and response to biotic and abiotic stresses. Despite their significance, a comprehensive analysis of the F-box gene family in Liriodendron chinense and other magnoliaceae species has not been reported. In this study, we report for the first time the identification of 144 full-length F-box genes in L. chinense. Based on specific domains and phylogenetic analyses, these genes were divided into 10 distinct subfamilies. We further analyzed their gene structure, conserved domain and chromosome distribution, genome-wide replication events, and collinearity. Additionally, based on GO analysis, we found that F-box genes exhibit functional specificity, with a significant proportion of them being involved in protein binding (GO:0005515), suggesting that F-box genes may play an important role in gene regulation in L. chinense. Transcriptome data and q-PCR results also showed that F-box genes are involved in the development of multiple tissues in L. chinense, regulate the somatic embryogenesis of Liriodendron hybrids, and play a pivotal role in abiotic stress. Altogether, these findings provide a foundation for understanding the biological function of F-box genes in L. chinense and other plant species.

Genome-Wide Identification and Abiotic-Stress-Responsive Expression of CKX Gene Family in Liriodendron chinense.

Liriodendron chinense is a tree species of the Magnoliaceae family, an ancient relict plant mainly used for landscaping and timber production due to its excellent material properties and ornamental value. The cytokinin oxidase/dehydrogenase (CKX) enzyme regulates cytokinin levels and plays an important role in plant growth, development, and resistance. However, too-high or too-low temperatures or soil drought can limit the growth of L. chinense, representing a key issue for research. Here, we identified the CKX gene family in the L. chinense genome and examined its transcriptional responses to cold, drought, and heat stresses. A total of five LcCKX genes, distributed on four chromosomes and divided into three phylogenetic groups, were identified across the whole L. chinense genome. Further analysis showed that multiple hormone- and stress-responsive cis-acting elements are located in the promoter regions of LcCKXs, indicating a potential role of these LcCKXs in plant growth, development, and response to environmental stresses. Based on existing transcriptome data, LcCKXs, especially LcCKX5, were found to transcriptionally respond to cold, heat, and drought stresses. Furthermore, quantitative reverse-transcription PCR (qRT-PCR) showed that LcCKX5 responds to drought stress in an ABA-dependent manner in stems and leaves and in an ABA-independent manner in roots. These results lay a foundation for functional research on LcCKX genes in the resistance breeding of the rare and endangered tree species of L. chinense.

Genome-Wide Analysis and Abiotic Stress-Responsive Patterns of COBRA-like Gene Family in Liriodendron chinense.

The COBRA gene encodes a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein (GAP), which plays an important role in cell wall cellulose deposition. In this study, a total of 7 COBRA-like (COBL) genes were identified in the genome of the rare and endangered woody plant Liriodendron chinense (L. chinense). Phylogenetic analysis showed that these LcCOBL genes can be divided into two subfamilies, i.e., SF I and II. In the conserved motif analysis of two subfamilies, SF I contained 10 predicted motifs, while SF II contained 4-6 motifs. The tissue-specific expression patterns showed that LcCOBL5 was highly expressed in the phloem and xylem, indicating its potential role in cellulose biosynthesis. In addition, the cis-element analysis and abiotic stress transcriptomes showed that three LcCOBLs, LcCOBL3, LcCOBL4 and LcCOBL5, transcriptionally responded to abiotic stresses, including cold, drought and heat stress. In particular, the quantitative reverse-transcription PCR (qRT-PCR) analysis further confirmed that the LcCOBL3 gene was significantly upregulated in response to cold stress and peaked at 24-48 h, hinting at its potential role in the mechanism of cold resistance in L. chinense. Moreover, GFP-fused LcCOBL2, LcCOBL4 and LcCOBL5 were found to be localized in the cytomembrane. In summary, we expect these results to be beneficial for research on both the functions of LcCOBL genes and resistance breeding in L. chinense.

AT-Hook Transcription Factors Show Functions in Liriodendron chinense under Drought Stress and Somatic Embryogenesis.

AT-hook motif nuclear localized (AHL) is a transcription factor that can directly induce plant somatic embryogenesis without adding exogenous hormones. One of its functional domains, the AT-hook motif, has a chromatin-modifying function and participates in various cellular processes, including DNA replication and repair and gene transcription leading to cell growth. Liriodendron chinense (Hemsl.) Sargent is an important ornamental and timber tree in China. However, its low drought-resistant ability further leads to a low natural growth rate of its population. Based on bioinformatics analysis, this study identified a total of 21 LcAHLs in L. chinense. To explore the expression pattern of the AHL gene family under drought and somatic embryogenesis, we performed a systematic analysis including basic characteristics, gene structure, chromosome localization, replication event, cis-acting elements and phylogenetic analyses. According to the phylogenetic tree, the 21 LcAHL genes are divided into three separate clades (Clade I, II, and III). Cis-acting element analysis indicated the involvement of the LcAHL genes in drought, cold, light, and auxin regulation. In the generated drought stress transcriptome, a total of eight LcAHL genes showed increased expression levels, with their expression peaking at 3 h and leveling off after 1 d. Nearly all LcAHL genes were highly expressed in the process of somatic embryogenesis. In this study, we performed a genome-wide analysis of the LcAHL gene family and found that LcAHLs take part in resistance to drought stress and the development of somatic embryos. These findings will provide an important theoretical basis for understanding of the LcAHL gene function.

Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress.

BACKGROUND: Liriodendron chinense (Lchi) is a tree species within the Magnoliaceae family and is considered a basal angiosperm. The too low or high temperature or soil drought will restrict its growth as the adverse environmental conditions, thus improving L. chinense abiotic tolerance was the key issues to study. WRKYs are a major family of plant transcription factors known to often be involved in biotic and abiotic stress responses. So far, it is still largely unknown if and how the LchiWRKY gene family is tied to regulating L. chinense stress responses. Therefore, studying the involvement of the WRKY gene family in abiotic stress regulation in L. chinense could be very informative in showing how this tree deals with such stressful conditions. RESULTS: In this research, we performed a genome-wide analysis of the Liriodendron chinense (Lchi) WRKY gene family, studying their classification relationships, gene structure, chromosomal locations, gene duplication, cis-element, and response to abiotic stress. The 44 members of the LchiWRKY gene family contain a significant amount of sequence diversity, with their lengths ranging from 525 bp to 40,981 bp. Using classification analysis, we divided the 44 LchiWRKY genes into three phylogenetic groups (I, II, II), with group II then being further divided into five subgroups (IIa, IIb, IIc, IId, IIe). Comparative phylogenetic analysis including the WRKY families from 17 plant species suggested that LchiWRKYs are closely related to the Magnolia Cinnamomum kanehirae WRKY family, and has fewer family members than higher plants. We found the LchiWRKYs to be evenly distributed across 15 chromosomes, with their duplication events suggesting that tandem duplication may have played a major role in LchiWRKY gene expansion model. A Ka/Ks analysis indicated that they mainly underwent purifying selection and distributed in the group IId. Motif analysis showed that LchiWRKYs contained 20 motifs, and different phylogenetic groups contained conserved motif. Gene ontology (GO) analysis showed that LchiWRKYs were mainly enriched in two categories, i.e., biological process and molecular function. Two group IIc members (LchiWRKY10 and LchiWRKY37) contain unique WRKY element sequence variants (WRKYGKK and WRKYGKS). Gene structure analysis showed that most LchiWRKYs possess 3 exons and two different types of introns: the R- and V-type which are both contained within the WRKY domain (WD). Additional promoter cis-element analysis indicated that 12 cis-elements that play different functions in environmental adaptability occur across all LchiWRKY groups. Heat, cold, and drought stress mainly induced the expression of group II and I LchiWRKYs, some of which had undergone gene duplication during evolution, and more than half of which had three exons. LchiWRKY33 mainly responded to cold stress and LchiWRKY25 mainly responded to heat stress, and LchiWRKY18 mainly responded to drought stress, which was almost 4-fold highly expressed, while 5 LchiWRKYs (LchiWRKY5, LchiWRKY23, LchiWRKY14, LchiWRKY27, and LchiWRKY36) responded equally three stresses with more than 6-fold expression. Subcellular localization analysis showed that all LchiWRKYs were localized in the nucleus, and subcellular localization experiments of LchiWRKY18 and 36 also showed that these two transcription factors were expressed in the nucleus. CONCLUSIONS: This study shows that in Liriodendron chinense, several WRKY genes like LchiWRKY33, LchiWRKY25, and LchiWRKY18, respond to cold or heat or drought stress, suggesting that they may indeed play a role in regulating the tree's response to such conditions. This information will prove a pivotal role in directing further studies on the function of the LchiWRKY gene family in abiotic stress response and provides a theoretical basis for popularizing afforestation in different regions of China.

Characterization of the Liriodendron Chinense MYB Gene Family and Its Role in Abiotic Stress Response.

Liriodendron chinense (Lchi) is a Magnoliaceae plant, which is a basic angiosperm left behind by the Pleistocene and mainly distributed in the south of the Yangtze River. Liriodendron hybrids has good wood properties and is widely used in furniture and in other fields. It is not clear if they can adapt to different environmental conditions, such as drought and high and low temperatures, and the molecular mechanisms for this adaptation are unknown. Among plant transcription factors (TFs), the MYB gene family is one of the largest and is often involved in stress or adversity response signaling, growth, and development. Therefore, studying the role of MYBTFs in regulating abiotic stress signaling, growth, and development in Lchi is helpful to promote afforestation in different environments. In our research, a genome-wide analysis of the LchiMYB gene family was performed, including the phylogenetic relationship tree, gene exon-intron structure, collinearity, and chromosomal position. According to the evolutionary tree, 190 LchiMYBs were divided into three main branches. LchiMYBs were evenly distributed across 19 chromosomes, with their collinearity, suggesting that segment duplication events may have contributed to LchiMYB gene expansion. Transcriptomes from eight tissues, 11 stages of somatic embryogenesis, and leaves after cold, heat, and drought stress were used to analyze the function of the MYB gene family. The results of tissue expression analysis showed that most LchiMYB genes regulated bark, leaf, bud, sepal, stigma, and stamen development, as well as the four important stages (ES3, ES4, ES9, and PL) of somatic embryogenesis. More than 60 LchiMYBs responded to heat, cold, and drought stress; some of which underwent gene duplication during evolution. LchiMYB3 was highly expressed under all three forms of stress, while LchiMYB121 was strongly induced by both cold and heat stress. Eight genes with different expression patterns were selected and verified by quantitative real-time PCR (qRT-PCR) experiments. The results suggested that these LchiMYBs may regulate Lchi growth development and resistance to abiotic stress. This study shows the cross-regulatory function of LchiMYBs in the growth and development, asexual reproduction, and abiotic resistance of Lchi. This information will prove pivotal to directing further studies on the biological function of Lchi MYBTFs in genetic improvement and abiotic stress response.

Integrative analysis of transcriptome and proteome revealed nectary and nectar traits in the plant-pollinator interaction of Nitraria tangutorum Bobrov.

BACKGROUND: Nitraria tangutorum is an important desert shrub that shows resistance to drought, salt and wind erosion stresses. It is a central ecological species in its area. Here, we have studied how N. tangutorum has adapted to achieve a successful reproduction strategy. RESULTS: We found that N. tangutorum is mainly pollinated by insects of the Hymenoptera, Diptera and Coleoptera orders. Nitraria tangutorum has very small flowers, with the nectary composed of secretive epidermal cells from which nectar is secreted, located within the inner petals. In addition, analyzing the transcriptome of four successive flower developmental stages revealed that mainly differentially expressed genes associated with flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction show dynamic expression. From the nectar, we could identify seven important proteins, of which the L-ascorbate oxidase protein was first found in plant nectar. Based on the physiological functions of these proteins, we predict that floral nectar proteins of N. tangutorum play an important role in defending against microbial infestation and scavenging active oxygen. CONCLUSIONS: This study revealed that N. tangutorum is an insect-pollinated plant and its nectary is composed of secretive epidermal cells that specialized into secretive trichomes. We identified a large number of differentially expressed genes controlling flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction. We suggest that proteins present in N. tangutorum nectar may have both an antibacterial and oxygen scavenging effect. These results provide a scientific basis for exploring how the reproductive system of N. tangutorum and other arid-desert plants functions.

The PIN gene family in relic plant L. chinense: Genome-wide identification and gene expression profiling in different organizations and abiotic stress responses.

The auxin efflux carrier PIN-FORMED (PIN) proteins are required for the polar transport of auxin between cells through their asymmetric distribution on the plasma membrane, thus mediating the differential distribution of auxin in plants, finally, affecting plant growth and developmental processes. In this study, 11 LcPIN genes were identified. The structural characteristics and evolutionary status of LcPIN genes were thoroughly investigated and interpreted combining physicochemical property analysis, evolutionary analysis, gene structure analysis, chromosomal localization, etc. Multi-species protein sequence analysis showed that angiosperm PIN genes have strong purification options and some functional sites were predicted about PIN protein polarity, trafficking and activity in L. chinense. Further qRT-PCR and transcriptome data analysis indicated that the long LcPINs have highly expressed from globular embryo to plantlet, and the LcPIN6a started upregulated in cotyledon embryo. The LcPIN3 and LcPIN6a are both highly expressed during the development of stamens and petals and the expression of LcPIN2 is related to root elongation, suggesting that they may play an important role in these processes. Experiment data indicates that LcPIN5 and LcPIN8 might play a key role in auxin transport in Liriodendron stems and leaves under abiotic stress. Analyzed the response of LcPIN genes to abiotic stress and as a basis for uncovering the biological role of LcPIN genes in development and adaption to adverse environments. This study provides a foundation for further genetic and functional analyses.

The Transcriptome of Cunninghamia lanceolata male/female cone reveal the association between MIKC MADS-box genes and reproductive organs development.

BACKGROUND: Cunninghamia lanceolata (Chinese fir), a member of the conifer family Cupressaceae, is one of the most popular cultivated trees for wood production in China. Continuous research is being performed to improve C. lanceolata breeding values. Given the high rate of seed abortion (one of the reasons being the failure of ovule and pollen development) in C. lanceolata, the proper formation of female/male cones could theoretically increase the number of offspring in future generations. MIKC MADS-box genes are well-known for their roles in the flower/cone development and comprise the typical/atypical floral development model for both angiosperms and gymnosperms. RESULTS: We performed a transcriptomic analysis to find genes differentially expressed between female and male cones at a single, carefully determined developmental stage, focusing on the MIKC MADS-box genes. We finally obtained 47 unique MIKC MADS-box genes from C. lanceolata and divided these genes into separate branches. 27 out of the 47 MIKC MADS-box genes showed differential expression between female and male cones, and most of them were not expressed in leaves. Out of these 27 genes, most B-class genes (AP3/PI) were up-regulated in the male cone, while TM8 genes were up-regulated in the female cone. Then, with no obvious overall preference for AG (class C + D) genes in female/male cones, it seems likely that these genes are involved in the development of both cones. Finally, a small number of genes such as GGM7, SVP, AGL15, that were specifically expressed in female/male cones, making them candidate genes for sex-specific cone development. CONCLUSIONS: Our study identified a number of MIKC MADS-box genes showing differential expression between female and male cones in C. lanceolata, illustrating a potential link of these genes with C. lanceolata cone development. On the basis of this, we postulated a possible cone development model for C. lanceolata. The gene expression library showing differential expression between female and male cones shown here, can be used to discover unknown regulatory networks related to sex-specific cone development in the future.

Physiological and transcriptomic analyses of brassinosteroid function in moso bamboo (Phyllostachys edulis) seedlings.

MAIN CONCLUSION: This study demonstrates that brassinosteroid is essential for seedling and shoot growth in moso bamboo. The shoot of moso bamboo is known to grow extremely fast. The roles of phytohormones in such fast growth of bamboo shoot remain unclear. Here we reported that endogenous brassinosteroid (BR) is a major factor promoting bamboo shoot internode elongation. Reducing endogenous brassinosteroid level by its biosynthesis inhibitor propiconazole stunted shoot growth in seedling stage, whereas exogenous BR application promoted scale leaf elongation and the inclination of lamina joint of leaves and scale leaves. Genome-wide transcriptome analysis identified hundreds of genes whose expression levels are altered by BR and propiconazole in shoots and roots of bamboo seedling. The data show that BR regulates cell wall-related genes, hydrogen peroxide catabolic genes, and auxin-related genes. Our study demonstrates an essential role of BR in fast growth bamboo shoots and identifies a large number of BR-responsive genes in bamboo seedlings.

An improved correlation method for amplitude estimation of gravitational background signal with time-varying frequency.

In the measurement of the gravitational constant G with angular acceleration method, the accurate estimation of the amplitude of the useful angular acceleration generated by source masses depends on the effective subtraction of the spurious gravitational signal caused by room fixed background masses. The gravitational background signal is of time-varying frequency, and mainly consists of the prominent fundamental frequency and second harmonic components. We propose an improved correlation method to estimate the amplitudes of the prominent components of the gravitational background signal with high precision. The improved correlation method converts a sinusoidal signal with time-varying frequency into a standard sinusoidal signal by means of the stretch processing of time. Based on Gaussian white noise model, the theoretical result shows the uncertainty of the estimated amplitude is proportional to σNT, where σ and N are the standard deviation of noise and the number of the useful signal period T, respectively.

Influence of tungsten fiber's slow drift on the measurement of G with angular acceleration method.

In the measurement of the gravitational constant G with angular acceleration method, the equilibrium position of torsion pendulum with tungsten fiber undergoes a linear slow drift, which results in a quadratic slow drift on the angular velocity of the torsion balance turntable under feedback control unit. The accurate amplitude determination of the useful angular acceleration signal with known frequency is biased by the linear slow drift and the coupling effect of the drifting equilibrium position and the room fixed gravitational background signal. We calculate the influences of the linear slow drift and the complex coupling effect on the value of G, respectively. The result shows that the bias of the linear slow drift on G is 7 ppm, and the influence of the coupling effect is less than 1 ppm.

Influence of temperature on period of torsion pendulum with a high-Q fused silica fiber.

Due to the high-Q fused silica fiber's extreme sensitivity to temperature change, the period estimation of torsion pendulum with high precision depends on the effective correction of the thermoelastic effect. In the measurement of G with the time-of-swing method, we analyze the complex relation between temperature and the pendulum's period and propose a developed method to find the shear thermoelasticity coefficient as well as isolate the influence of temperature on period alone. The result shows that the shear thermoelasticity coefficient is 101(2) × 10(-6)/°C, the resultant correction to Δ(ω(2)) is 9.16(0.18) ppm, and the relative uncertainty to G is less than 1 ppm.