Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla

doi:10.21203/rs.3.rs-3021067/v1

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Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla

https://doi.org/10.21203/rs.3.rs-3021067/v1

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published 08 Feb, 2024

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Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the GATA TF family in U. rhynchophylla has not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIA content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content.

Uncaria rhynchophylla

Genome

GATA transcription factor

Terpenoid indole alkaloids biosynthesis

Gene expression profiles

Hypertension is one of the most common chronic diseases in many countries, and is a risk factor for many diseases such as heart disease, and diabetes mellitus (Kearney et al., 2005). China has the largest population with hypertension in the world and recent surveys estimated that almost one third of Chinese adults have hypertension, so possible treatments for hypertension have received significant attention (Lewington et al., 2016; Lu et al., 2017; Wei et al., 2021). U. rhynchophylla is an evergreen vine plant classified into the Rubiaceae family and contains many terpenoid indole alkaloids (TIAs), including isocorynoxeine, corynoxeine, isorhynchophylline, and rhynchophylla, that are often used to treat hypertension, epileptic seizures, and Alzheimer’s disease (Ndagijimana et al., 2013; Tang et al., 2017; Zeng et al., 2021). The biosynthesis pathway of U. rhynchophylla TIAs is complex, and several steps still require clarification (Guo et al., 2014). In general, the terpene backbone of TIAs originate from the 2-C-methyl-D-erythritol 4-phosphate (MEP) and the mevalonate (MVA) pathways. In the iridoid pathway, Geranyl-PP, is converted to geraniol under the catalysis of geranyl diphosphate diphosphatase (GES) (Suttipanta et al., 2011), then geraniol is then hydroxylated to 8-hydroxygeraniol under the catalysis of geraniol 8-hydroxylase (G8H) (Wang et al., 2023). 8-hydroxygeraniol is then oxidated to 8-oxogeranial by 8-hydroxygeraniol dehydrogenase (8-HGO) (Partridge et al., 2021). The cyclization of 8-oxogeranial to form iridodial catalyzed by iridodial synthase (IS), is then followed by the conversion to 7-deoxyloganetic acid by a two-step oxidation of iridodial oxidoreductase (IO) (Miettinen et al., 2014). 7-deoxyloganetic acid is then converted to 7-deoxyloganic acid under the action of a 7-deoxyloganetic acid glucosyltransferase (7-DLGT) (Cui et al., 2015), and catalyzed to the formation of loganic acid through a 7-deoxyloganic acid hydroxylase (7-DLH) (Asada et al., 2013). Finally, secologanin, a precursor to the U. rhynchophylla TIAs, is then catalyzed by secologanin synthase (SLS) (Irmler et al., 2000). In the indole pathway, tryptamine as an another precursor, is formed from L-Tryptophan under the action of the L-tryptophan decarboxylase (TDC) (Shao et al., 2023). Strictosidine is a key rate-limiting enzyme in the synthesis of TIAs, which is formed by the condensation of tryptamine and secologanin under the catalysis of strictosidine synthase (STR). The U. rhynchophylla TIAs are then formed via a series of reactions that have not been elucidated (Fig. 1). The U. rhynchophylla genome was recently sequenced by Oxford Nanpore technology, with a genome size of 627 Mb and a contigN50 of 1.8 Mb (not published). This genome sequence provides a large genetic resource for the analysis of the U. rhynchophylla TIA pathways.

During the life of a plant, many factors including external and internal factors affect its growth and development (Feng et al., 2020). Transcription factors (TFs), as one of the internal factors, are important regulators of developmental processes in higher plants. The GATA family is a large group of transcription factors that can regulate plant growth and development, secondary metabolism, and signal transduction (Moon et al., 2022; Schwechheimer et al., 2022). GATA TFs contain one or two highly conserved type-IV zinc fingers (C-X₂-C-X_{17 − 20}-C-X₂-C) and a DNA binding region that recognizes the sequence (T/A) GATA (A/G) in the target promoter region (Zhang et al., 2019). Most GATA TFs contain a single C-X₂-C-X₁₈-C-X₂-C motif, however some GATA TFs include more than two zinc-finger motifs or zinc-finger loops of 20 residues in plants (Reyes et al., 2004). The first plant GATA gene NTL1 was isolated from Nicotiana tabacum (Du et al., 2022), but using analysis based on whole genome sequences all GATA TF members have been identified in Arabidopsis thaliana (30), Oryza sativa (29), and Triticum aestivum (79) (Du et al., 2022; Zhang et al., 2015). According to the multiple sequence-based alignments, phylogenetic relationships, conserved motifs, and gene structure, the GATA family can be divided into four subgroups, I, II, III, IV (Feng et al., 2022).

A number of specific GATA TFs in different plant species have been shown to be important regulators of growth and development. In A. thaliana, the BME3 GATA mRNA accumulated in seeds during the cold stratification, and seeds showed deeper dormancy when the gene was knocked out, implying that the BME3 GATA is a positively regulator of seed germination (Liu et al., 2005). Overexpression of OsGATA8 displayed higher accumulation and photosynthetic efficiency in O. sativa seedlings compared to wild-type and knockout plants (Nutan et al., 2020). In addition, GATA TFs have also been shown to be involved in abiotic and biotic responses. In Solanum lycopersicum, overexpressing SlGATA17 in tomatoes enhanced drought resistance and improved the activity of the phenylpropanoid biosynthesis pathway (Zhao et al., 2021). Several GmGATA genes exhibited transcriptional upregulation or downregulation in response to low nitrogen stress, and GmGATA44 and GmGATA58 were thought to be potentially involved in the regulation of nitrogen metabolism in soybean (Zhang et al., 2015). GATA TFs binding motifs have been found in many regulatory regions of light-responsive genes. In A. thaliana, two GATA genes were strongly regulated by light and expressed in photosynthetic organs, and several GATA genes have been demonstrated to be responsive to light, dark, and changes in circadian rhythms (Manfield et al., 2007). In Catharanthus roseus, overexpression or silencing of CrPIF1 in C. roseus seedlings altered the expression of CrGATA1 as well as the expression of genes associated with the vindoline pathway of plants grown in the dark, indicating that CrPIF-GATA potentially governs light-mediated biosynthesis of the TIA pathways (Liu et al., 2019). Light intensity is an important key environmental variable affecting TIAs biosynthesis and accumulation, for example, the content of rhynchophylline and isorhynchophylline significantly increased at 20% light intensity after 7 and 21 days, compared with 100% light intensity, and some genes involved in iridodial pathway were found to be up-regulated under 7 and 21 days of shade treatment in U. rhynchophylla (Wang XH et al., 2022). Thus, light may critically affect the regulation of GATA and TIAs of U. rhynchophylla.

From the analysis of the U. rhynchophylla genome, we systematically identified and characterized the entire GATA TF family, consisting of 25 genes. Altering the light conditions under which U. rhynchophylla seedlings were grown, resulted in the altered accumulation of four kinds of U. rhynchophylla alkaloids. Gene expression analysis conducted on all 25 UrGATA genes and 19 genes involved in TIAs pathway, under the different light growth conditions, identified potential candidate UrGATA genes that could participate in the light responsive regulation of the TIA biosynthesis pathways. Our results provide new insights into the function of UrGATA genes under different light conditions.

2.1 Identification and characterization of GATA genes in U. rhynchophylla

The hidden Markov model of the GATA domain (PF00320) stemming from the Pfam database (http://pfam.xfam.org) was used to identify potential members of the U. rhynchophylla GATA gene family (Kim et al., 2021b). NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and Expasy (https://prosite.expasy.org/) were used to verify the integrity of the GATA domain (Yao et al., 2022). In addition, the online website ExPASy (https://web.expasy.org/protparam/) was used to calculate the physical and chemical properties of UrGATA proteins. Subcellular localization of UrGATA proteins was predicted using Plant-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/)

2.2 Multiple sequence alignment and construction of phylogenetic tree

The U. rhynchophylla genome database was prepared to identify UrGATA family members. The GATA protein sequences from A. thaliana and O. sativa were downloaded from TAIR database (https://www.arabidopsis.org/index.jsp) and the Genome Annotation Project (http://rice.plantbiology.msu.Edu/cgi- bin /ORF_ info page.cgi). MAFFT software was used for multiple sequence alignment of UrGATA proteins (Katoh et al., 2019). MEGA 7.0 was used to construct the phylogenetic tree among U. rhynchophylla, A. thaliana and O. sativa using neighbor-joining method with 1000 bootstrap (Mu et al., 2023).

2.3 The analysis of conserved motifs and gene structures

The online website MEME v5.1.1 (https://meme-suite.org/meme/) was employed to predict the conserved motifs of UrGATA proteins, with the following parameters: maximum number of motifs = 8; the length of motifs, ranging from 20 to 50 (Mu et al., 2023). The DNA and CDS sequences of UrGATA gene family were screened from U. rhynchophylla genome, and gene structures were visualized by TBtools v1.108 software (Jiang et al., 2021).

2.4 Chromosomal location, gene duplication and collinearity analysis

The method for locating UrGATA genes on 22 chromosomes of U. rhynchophylla was similar to that of WRKY genes in Siraitia siamensis (Mu et al., 2023). Gene duplication events were employed by one step (MCScanX) in TBtools with the following parameters: CPU for BlastP:8; E-value:1e^− 10 (Wang et al., 2012). TBtools software was used to build collinearity analysis between U. rhynchophylla and four other species (A. thaliana, O. sativa, C. canephora, and C. roseus) (Shi et al., 2022).

2.5 Cis-acting element analysis and RNA-seq expression patterns

The 2000 bp upstream promoter region of UrGATA genes was extracted by TBtools software (GTF/GFF3 Sequences extractor), and the online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict cis-acting elements in the promoter regions of UrGATA genes (Lescot et al., 2002). The transcript per kilobase per million mapped reads (TPM) value of UrGATA family members in four different tissues were acquired from transcriptome database of U. rhynchophylla. The relative expression level of UrGATA genes in the form of a heat map was obtained by TBtools software (Heatmap Illustrator).

2.6 Protein interaction network of UrGATA family members

The protein interaction network of UrGATAs was constructed from the online website STRING (http://string-db.org/), which is an open-source software for predicting and visualizing complex networks (Wang et al., 2020). These interactions originated from manuscripts concerning experimental validation of physical interactions and reactions related with signal transduction (Jiang et al., 2021).

2.7 Plant material and different light treatments

The U. rhynchophylla plants used in this research were collected from Guangxi Botanical Garden of Medicinal Plants, Nanning City, Guangxi Province, China (108.19E;22.49N), and identified by Prof. Shugen Wei. The growth condition of tissue seedlings was reported previously (Shao et al., 2023). Two months later, the tissue seedlings (six plants in each group) were treated with white light (150 µmol/m²/s), red light (35 µmol/m²/s), and 20% light intensity (30 µmol/m²/s). The growth conditions were consistent among three groups except for differences in light. Leaves were collected from three independent plants as three biological replicates on days 0, 1, 4, 7, 14, and 21 after treatments. The collected leaves were divided into two parts. One part was instantly frozen in liquid nitrogen and then stored at -80°C refrigerator to extract total RNA. The other part was used to test the content of four kinds of U. rhynchophylla alkaloids.

2.8 The analysis of four U. rhynchophylla alkaloids accumulation

The U. rhynchophylla leaves were dried at 60℃ and then crushed, screened through 60-micron mesh and collected. The contents of four U. rhynchophylla alkaloids were analyzed by high performance liquid chromatography (HPLC, Sinopharm Chemical Reagent, Shanghai, China). Each sample was weighed and then 1 mL of 80% methanol (HPLC grade) was added to extract alkaloids. The mixture was sonicated in an ultrasonic bath (Ningbo Scientz Biotechnology Co. Ltd) for 60 min, followed by centrifugation at 12,000 rpm for 5 min, and the supernatant was filtered through a nitrocellulose filter (0.22 µm). Four TIAs ingredients, isocorynoxeine, corynoxeine, isorhynchophylline, and rhynchophylline, were detected using the following methods: GL-Sciences GL-C18 column (250 mm × 4.6 mm, 5 µm), using 0.2% ammonia (phase A) ~ acetonitrile (phase B) as the mobile phase of the system, and using gradient elution procedure: 0 ~ 40 min, 28 ~ 55% B; 40 ~ 60 min, 55 ~ 63% B; 60 ~ 60 min, 63 ~ 28% B; 60 ~ 70 min, 28 ~ 28% B. The detection wavelength was set at 254 nm, the chromatographic column temperature 30℃, the flow rate 1.0 mL/min, and the injection volume 20 µL. The standard products were accurately weighed: isocorynoxeine (1 mg), corynoxeine (1 mg), isorhynchophylline (1 mg), and rhynchophylline (1 mg), and then dissolved in 80% methanol. These were then diluted to 5 ml to prepare the stock solution. These stock solutions were diluted to make a series of concentration gradient solutions for the standard curve.

2.9 Total RNA extraction, cDNA synthesis, and RT-qPCR conditions

Leaf samples (50–100 mg) were fully ground in the liquid nitrogen, and total RNA extracted using the SteadyPure Plant RNA extraction Kit (Accurate Biology, Hunan, China). The concentration and purity of RNA were measured using Micro Drop (BIO-DL, Shanghai, China), and the integrity of RNA was checked by 1% agarose gels. Total RNA (1 µg) was used for reverse transcription with Evo M-MLV RT Mix Kit (Accurate Biology, Hunan, China). Genes involved in TIA pathways were obtained from the U. rhynchophylla genome sequence database. Specific primer pairs were designed using Beacon Designer 7.0 software and the primers used for this study are shown in Table S1. The reaction was carried out in 96-well plates with an ABI 7300 (ABI, America). The reaction system and procedure were previously reported (Mu et al., 2023). All qRT-PCR analysis were performed with three biological replicates. The relative expression was calculated with the reference gene, SAM, using the 2^−∆∆CT method. The statistical tests were used GraphPad Prism 9.0 software. Correlation analysis between content and gene expression was performed by OmicStudio online website (https://www.omicstudio.cn/), significant difference was inspected at significance levels of 0.05, 0.01, and 0.001.

3.1 Identification and characteristics of U. rhynchophylla GATA TFs

In this study, a total of 25 GATA genes were identified from U. rhynchophylla genome (BioProject: PRJNA931770) and were named as UrGATA1-UrGATA25 corresponding to their chromosomal location. The characteristics of 25 UrGATA genes, including the number of amino acids, molecular weight, theoretical pI (pI), instability index, and subcellular localization are presented in Table S2. The predicted lengths of the U. rhynchophylla GATA TFs ranged from 134 (UrGATA15) to 461 (UrGATA11) amino acid, and the molecular weights ranged from 15.15 kDa (UrGATA15) to 50.97 kDa (UrGATA11). The predicted pI values ranged from 5.33 (UrGATA24) to 10.81 (UrGATA10). All UrGATA proteins were considered as unstable proteins. Subcellular localization prediction indicated all 25 GATA proteins were found in the nucleus.

3.2 Phylogenetic analysis and multiple sequence alignment of UrGATA

A neighbor-joining tree was constructed using MEGA 7.0 for multiple sequence alignment of full length GATA proteins from U. rhynchophylla, A. thaliana, and O. sativa with 1000 bootstrap replications to analyze phylogenetic relationships (Fig. 2). According to the GATA classification of A. thaliana, and O. sativa, UrGATA proteins were divided into four subgroups, I, II, III, IV (Fig. 2, Fig. 3a). Subgroup I with 13 UrGATAs members was the largest group (UrGATA2, UrGATA6, UrGATA8, UrGATA11-14, UrGATA17, UrGATA19-21, UrGATA23-24). Nine UrGATAs were placed in subgroup II (UrGATA1, UrGATA3-5, UrGATA9, UrGATA15, UrGATA18, UrGATA22, UrGATA25), and subgroup III had only one UrGATA designated UrGATA16. UrGATA7 and UrGATA10 were grouped into subgroup IV.

3.3 Conserved motifs and gene structure analysis of UrGATA gene family

To better analyze the diversity of UrGATA proteins, conserved protein motifs were predicted using the online website MEME. In total, eight conserved motifs were identified (motif 1–8) (Fig. 3b, Fig. 3d, Table S3). Motif 1 was detected in all UrGATA proteins. Motifs 2, 3, 5, 6, 7, and 8 were only found in subgroup I, and motif 4 was only observed in subgroup II.

To gauge the genetic structural diversity of UrGATA genes, the exon/intron analysis of DNA sequences of UrGATA genes was performed. As is shown in Fig. 2c, the number of introns in UrGATA genes varied from one to six (twelve with one intron, nine with two introns, one with three introns, two with four introns, one with one intron), in which UrGATA16 contained the greatest number of predicted introns (n = 6). In general, UrGATA members in the same subgroup displayed similar gene structures, corresponding to the phylogenetic tree analysis of subgroups. Similar to the previous reports of A. thaliana, O. pumila, and O. sativa, 25 UrGATA proteins belonging to different subgroups contained the same conserved domain, only UrGATA16 belonging to subgroup III had the C-X₂-C-X₂₀-C-X₂-C structure, the other UrGATAs had the conserved domain, C-X₂-C-X₁₈-C-X₂-C (Fig. 4).

3.4 Chromosomal distribution and collinearity analysis of UrGATA gene family

The location map of 25 UrGATA genes in the genome of U. rhynchophylla are shown in the Fig. 5. UrGATA genes were most abundant on Ur_chr7 (UrGATA7-9) and Ur_chr16 (UrGATA17-19). Six chromosomes (Ur_chr1, Ur_chr3, Ur_chr10, Ur_chr12, Ur_chr13, Ur_chr21) had no UrGATA genes and the other chromosomes contained 1–2 UrGATA genes. Synthetic blocks within the U. rhynchophylla genome were analyzed to identify the relationship between UrGATA genes and gene replication events (Fig. 6). The results showed that no tandem duplication gene pairs were identified among 25 UrGATA genes, while 28 gene pairs of fragment repeats were detected between fifteen chromosomes. According to previous studies, gene replication is a key reason for the gene expansion (Doksani, 2019). Therefore, some UrGATA genes might have been generated by gene duplication.

To obtain a more comprehension understanding of the evolution of the UrGATA family, synteny analysis comparing U. rhynchophylla with three dicotyledonous plant species (A. thaliana, C. canephora, and C. roseus) and one monocotyledonous species (O. sativa) were constructed (Fig. 7, Table S4). UrGATA genes showed different collinearity relationship with A. thaliana (48), C. roseus (45), C. canephora (39), O. sativa (11), with UrGATA genes having a comparatively greater similarity relationship with that in A. thaliana.

3.5 Cis-acting elements analysis of UrGATA gene family

To gain further insight into the potential functions of GATA TFs in U. rhynchophylla, 2000-bp regulatory upstream region of translation initiation site were extracted to identify cis-acting elements using the PlantCARE. Cis-acting elements were divided into three categories, plant growth and development, phytohormone responsive, and stress responsive boxes. 25 UrGATA TFs contained at least one cis-acting element in the promoter region (Fig. 8). The elements related to plant growth and development (266), containing light responsive and development-related elements were found in the promoter region. The elements, G-box (69), Box4 (61), GT1- motif (26), GATA-motif (17), and Sp1 (7), are associated with light responses. There were also elements corresponded with growth, such as O₂-site (13), and CAT-box (11). Phytohormone responsive elements for abscisic acid (ABA) (58), methyl jasmonate (MeJA) (50), salicylic acid (SA) (26), gibberellin (GA) (16), auxin (13) were also identified in the promoters of UrGATA genes. Abiotic and biotic stress-related elements were also found in UrGATA promoter regions, with antioxidant response (ARE) (58), drought (MBS) (15), low temperature (LTR) (14), wound responsive (WUN-motif) (2) boxes, identified. These prediction results indicated that several cis-acting elements may regulate UrGATA genes during the growth and phytohormone response.

3.6 Protein network interaction and expression patterns of UrGATA genes

The expression patterns of 25 UrGATA genes in four different tissues (root, stem hook, leaf, flower) were evaluated using the transcriptome database of U. rhynchophylla (Fig. 9). Among all 25 UrGATA genes, 64% UrGATA genes (16/25) were expressed in all samples (TPM > 0), whereas 20% UrGATA genes (5/25) were not expressed in any tissues. Furthermore, 40% (10/25) UrGATA genes displayed highest expression levels in the flower, followed by 16% (4/25) of UrGATA genes that had the highest expression levels in stem hook and root, and 8% (2/25) of UrGATA genes had the highest expression levels in roots. These results showed that the expression patterns of 25 UrGATA genes were diverse in different tissues, and therefore, likely performing different functions in these tissues.

The software STRING was used to build a protein-protein interaction network of 13 UrGATA proteins based on the transcriptome database of the four different tissues. In the protein interaction network (Fig. 10), the darker the color, the greater the number of interactions with other GATA proteins. Among the 13 UrGATA proteins, five UrGATA proteins belonged to subgroup I, five UrGATA proteins belonged to subgroup II, one UrGATA protein belonged to subgroup III, and the remaining were grouped into subgroup IV. UrGATA4, UrGATA5, UrGATA17, UrGATA22, UrGATA24, as five key nodes in protein interaction network, have different intensities of interaction with other UrGATA proteins. The results showed that these UrGATA proteins might play a key role in the whole GATA regulation process, especially UrGATA4.

3.7 The accumulation of four TIAs in U. rhynchophylla under different lights

The determine whether altered light treatments could affect TIA accumulation in tissue culture grown U. rhynchophylla seedlings, four TIAs, corynoxeine, isocorynoxeine, isorhyncophylline, and rhyncophylline, were measured at six time points (0d, 1d, 4d, 7d, 14d, 21d) under three different conditions: normal light (CK), 20% light and red light (see Material and methods). As is shown in the Fig. 11, for corynoxeine, neither treatment resulted in significant changes until 7d, where it was observed that red light resulted in increased accumulation from 7d to 21d. Whereas, 20% light treatment produced less corynoxeine at 7d and 14d, but produced higher levels by 21d. For isocorynoxeine red light treatment resulted in steady increases in production over the course of the experiment, whereas 20% light, had higher content at 7d and 21d only. The results for isocorynoxeine were complex, as red light resulted in increased content only at 7d and 21d, while 20% light initially caused a decrease in content at 4d, but then resulted in significant increases at all later time points (7d, 14d, and 21d). Analysis of rhyncophylline content, found that both red light and 20% light treatments resulted in increases from 7d onwards to 21d.

These results indicate that responses to the different light treatments were treatment and TIA specific, but both treatments could significantly alter the content of the four measured TIA during the experiment. In general, the light treatments resulted in increased levels compared to the control that occurred at later time points (by 7d).

3.8 Expression patterns of UrGATA genes and key enzyme genes under different lights

The expression profiles of 25 UrGATA genes and key TIA enzyme genes from leaf tissue under normal light, 20% light and red light conditions were examined using RT-qPCR, and expression heat maps generated to display expression profiles (Fig. 12a, Fig. 12b). Several UrGATA genes (UrGATA1, UrGATA9, UrGATA14-15, UrGATA18, UrGATA20-21, and UrGATA24) were not expressed in leaf tissue, which is consistent with the transcription sequencing data (Fig. 9, Fig. 12a). However, several UrGATA genes (UrGATA2, UrGATA4-5, UrGATA11, UrGATA13, UrGATA17, and UrGATA22) were up-regulated under the red light during 21d treatment, compared to CK. Whereas, most UrGATA genes were down-regulated under 20% light condition, with only three UrGATA genes (UrGATA7, UrGATA8, UrGATA23) found to have increased expression.

For key enzyme genes involved in the TIA pathways, UrG8H, Ur7DLGT, UrAS, UrAnPRT, UrTSA, UrTDC, UrSTR, and UrSGD were significantly up-regulated by red light, while the expression of Ur8HGO and Ur7DLH were increased but not significantly compared to the CK. Interestingly, UrGES, Ur8HGO, UrIO, Ur7DLH, UrLAMT, UrSLS, UrTSA, and UrTSB were highly up-regulated under the 20% light treatment compared to the CK (Fig. 12b).

Correlation analysis between UrGATA genes and key enzyme genes were analyzed (Fig. 13). The results showed that UrGATA7 and UrGATA8 were positively correlated with key enzyme genes analyzed, showing high expression under the different light treatments. Among these, UrAS, UrTDC, and UrG8H showed strong correlations (p < 0.05, r > 0.8) with UrGATA8, and UrGATA7 had significant correlations with UrSGD, UrLAMT, UrIO, and Ur7DLH (p < 0.05, r > 0.8). Moreover, UrGATA23 was positively correlated with the expression of UrGES, (Pearson coefficients 0.87). These results indicate that GATA genes and key enzyme genes could share similar regulation by the light treatments, and may therefore, regulate the expression of key enzyme genes that result in an increase TIA content.

To predict whether UrGATA transcription factors can bind to cis-acting elements in the promoter region of key enzyme genes, 2.0Kb promoter region of these important enzyme genes involved in TIAs pathway of U. rhynchophylla were predicted using the online website PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Based on the prediction, the GATA cis-acting elements were found in the promoter region of UrGES, UrG8H, Ur8HGO, UrSLS,UrTSB, and UrTDC. Therefore, providing support for a possible method by which these key enzyme genes might be regulated by UrGATA TFs.

U. rhynchophylla is rich in a variety of TIAs that can be used in the treatment of hypertension and Alzheimer's disease (Ndagijimana et al., 2013; Pan et al., 2018; Zeng et al., 2021). Therefore, U. rhynchophylla has received widespread attention that resulted in the completion of the sequencing of the U. rhynchophylla genome by ONT. A major aim of U. rhynchophylla research is to understand and alter the TIA biosynthetic pathways through genetic engineering to increase TIA accumulation. For instance, overexpression of UrSTR1 and UrSTR5 elevated the bioactive ingredients isorhynchophylline and rhynchophylline, while RNAi silencing of UrSTR1 and UrSTR5, decreased UR-TIAs accumulation (Jiang et al., 2023). The identification and regulation of transcription factors associated with TIA biosynthesis pathways have been a major area of study. By sequencing the transcriptome of U. rhynchophylla, 1065 transcription factors were initially identified, that were classified into 55 TF families, with two TFs, HY5 and PIFs, found to be involved in light signaling pathway, as they showed different expression levels under reduced light treatment (Wang XH et al., 2022). Using established and optimized systems for the extraction and transfection of U. rhynchophylla protoplasts, it was found that the nuclear located transcription factor UrWRKY37 could bind to the W-box in the promoter region of UrTDC and activate its expression (Shao et al., 2023). Despite these successes complete bioinformatics analysis of transcription factor family based on genome-wide and their regulation of TIAs biosynthesis pathways is still lacking.

Transcription factors are a class of proteins that can bind to DNA and affect plant growth and development by regulating the transcription initiation of target genes (He et al., 2021). GATA transcription factors are one of the most important transcription regulatory factors in higher plants, that help regulate plants growth and response to various biotic and abiotic stresses (Hussain et al., 2021; Jiang et al., 2021), and have been widely studied in a series of plants including A. thaliana (Kim et al., 2021a), wheat (Feng et al., 2022), Capsicum annuum (Yu et al., 2021), Fagopyrum tataricum (Yao et al., 2022)d pumila (Shi et al., 2022). This study is the first comprehensive analysis of the GATA transcription factor family in U. rhynchophylla. Based on genome analysis, 25 UrGATA TFs were identified and designated UrGATA1-UrGATA25 on the basis of their location on the 22 chromosomes, and classified into four subgroups, consistent with A. thaliana (Kim et al., 2021a). The GATA TFs in the subgroup III have a zinc finger domain, consisting of a C-X₂-C-X₂₀-C-X₂-C structure, comprising 20 residues. The remaining three subgroups have a C-X₂-C-X₁₈-C-X₂-C structure with 18 residues. Subfamily III is distinguished by the presence of CCT and TIFY domains, as represented by UrGATA16. The CCT domain was first discovered in the A. thaliana Constans protein, which plays a crucial role in hypocotyl and root growth, and regulates the flowering process (Suárez-López et al., 2001). In addition, the family contained the TIFY conserved domain (Sun et al., 2021), which is divided into four subfamilies (TIFY, JAZ, ZML, and PPD) based on the specific types of additional domains it contains. Many studies have shown that JAZ proteins are an important regulator in jasmonic acid signal transduction pathway (Thireault et al., 2015).

Conserved motifs analysis revealed that all UrGATA proteins possessed motif 1, which might be a key functional element retained in the evolutionary process (Mu et al., 2023). Specific motifs were identified in other subgroups. For example, motif 3, motif 5, and motif 7 were exclusively present in subgroup I, whereas motif 4 was solely detected in subgroup II. These findings suggest that while certain motifs in the GATA gene family of U. rhynchophylla are highly conserved, that novel and specific species motifs might have unique functions in certain plant species. Further detailed investigations are required to elucidate the roles of these newly evolved motifs. Gene duplication events, including tandem, segmental, and whole genome duplication, are important for the acquisition of new genes and the expansion of gene families in organisms (Magadum et al., 2013). Among the 25 UrGATA genes, no gene pairs showed tandem duplication, however 28 gene pairs of fragment repeats were detected, that indicate that only segmental duplications are likely to contribute to expansion of UrGATA gene family (Wang C et al., 2022). Homology of the GATA gene of U. rhynchophylla with those of A. thaliana, O. sativa, C. canephora, and C. roseus were analyzed. Remarkably, the AtGATA2 (AT3G60530) (homolog of UrGATA16) has been reported that to be an important regulator of photoresponsive gene expression A. thaliana and plays a key role in the light signaling pathway (Luo et al., 2010). Furthermore, AtGATA1 (AT3G24050) (homolog of UrGATA22) could bind to the GATA cis-acting element in the promoter region of the GAPA gene, which is regulated by light (Jeong and Shih, 2003). The homologous GATA-type transcription factor from A. thaliana (AT5G56860) (homolog of UrGATA3), GNC, responds to auxin and gibberellin signals to control the plant growth and development (Richter et al., 2013). In a recent study, AtGATA12 (AT5G25830) (homolog of UrGATA12) was downregulated in response the GA treatment, and its expression levels decreased in dry storage and cold stratification of seeds circumstances (Ravindran et al., 2017). According to these results, it is possible that the corresponding UrGATA gene may also have similar functions, but this requires further research.

The expression levels of most of the key enzyme genes involved in TIAs biosynthesis pathway were found to be significantly increased both in response to 20% light and red light 21d treatment. UrGATA2, UrGATA4-5, UrGATA8, UrGATA11, UrGATA13, UrGATA17, UrGATA22 were significantly increased under 21d of red light compared to the control, indicating that these UrGATA genes might be mainly regulated by red light. However, only three UrGATA genes, UrGATA7, UrGATA8, and UrGATA23 were significantly increased under 21d of 20% light treatment compared to the control. UrGATA8 showed positive correlations with UrAS, UrTDC, and UrG8H, whereas UrGATA7 had positive significant correlations with UrSGD, UrLAMT, UrIO, and Ur7DLH. These results indicate that UrGATA7 and UrGATA8 have similar expression profiles to these key enzyme genes, implying that they may regulate their expression leading to increased accumulation of the four TIAs measured. Similar co-expression profiles have been reported previously, as in C. roseus, expression of CrGATA1 and five genes involved in vindoline pathway decreased coincidently with dark treatment, resulting in the significant decrease of vindoline content (Liu et al., 2019). In O. pumila, OpGATA7 showed positive association with genes involved in CPT biosynthesis pathway, implying that key enzyme genes had a strong possibility of being regulated (Shi et al., 2022).

In this research, the UrGATA TF family in U. rhynchophylla was comprehensively identified using the complete genome sequence of U. rhynchophylla. In total, 25 UrGATA genes, were identified and classified. Altering either the light quality or intensity that U. rhynchophylla seedling were grown under, resulted in the altered expression of many UrGATA genes as well as the content of four important TIAs. Several UrGATA genes expression profiles closely correlated with the expression of key TIA pathway enzyme genes, emphasizing the possible functions of UrGATA genes in the regulation of TIAs biosynthesis in U. rhynchophylla. This research provides a novel insight into the function and characteristics of UrGATA genes and lays a foundation for understanding the mechanism regulating TIAs biosynthesis pathway in U. rhynchophylla.

Funding

This work was supported by the National Natural Science Foundation of China (No.32270398), the Project of Hunan Natural Science Foundation (2022JJ30305), the Key Scientific Research Fund of Hunan Provincial Education Department (21A0138), the Horticulture’s Open program of Hunan Agricultural University (2021YYXK002), Postgraduate Scientific Research Innovation Project of Hunan Province (No. CX20220689) and Postgraduate Scientific Research Innovation Project of Hunan Agriculture University (No. 2022XC062).

Competing interests

The authors declare no conflict of interest

Authors’ contributions

QT and DTM initiated the project, helped to conceive the study, drafted the manuscript, and participated in the design and coordination. YYS, YZ and LY conceived the study, participated in experiments and data analysis, and IWW and DYQ participated in data analysis and manuscript revision. YZ, LNZ, XHL, LL and JLH also participated in experiments. All authors read and approved the final manuscript.

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Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla

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Journal Publication

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Abstract

Figures

1. Introduction

2. Material and methods

2.1 Identification and characterization of GATA genes in U. rhynchophylla

2.2 Multiple sequence alignment and construction of phylogenetic tree

2.3 The analysis of conserved motifs and gene structures

2.4 Chromosomal location, gene duplication and collinearity analysis

2.5 Cis-acting element analysis and RNA-seq expression patterns

2.6 Protein interaction network of UrGATA family members

2.7 Plant material and different light treatments

2.8 The analysis of four U. rhynchophylla alkaloids accumulation

2.9 Total RNA extraction, cDNA synthesis, and RT-qPCR conditions

3. Results

3.1 Identification and characteristics of U. rhynchophylla GATA TFs

3.2 Phylogenetic analysis and multiple sequence alignment of UrGATA

3.3 Conserved motifs and gene structure analysis of UrGATA gene family

3.4 Chromosomal distribution and collinearity analysis of UrGATA gene family

3.5 Cis-acting elements analysis of UrGATA gene family

3.6 Protein network interaction and expression patterns of UrGATA genes

3.7 The accumulation of four TIAs in U. rhynchophylla under different lights

3.8 Expression patterns of UrGATA genes and key enzyme genes under different lights

4 Discussion

5 Conclusion

Declarations

References

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