Identification and expression analysis of CCCH gene family and screening of key low temperature stress response gene CbuC3H24 and CbuC3H58 in Catalpa bungei.

Catalpa bungei, a tree indigenous to China, is renowned for its superior timber quality and as an ornamental in horticulture. To promote the cultivation of C. bungei in cold regions and expand its distribution, enhancing its cold tolerance is essential. The CCCH gene family is widely involved in plant growth, development, and expression under stress conditions, including low-temperature stress. However, a comprehensive identification and analysis of these genes have not yet been conducted. This study aims to identify key cold-tolerance-related genes within the CCCH gene family of C. bungei, providing the necessary theoretical support for its expansion in cold regions. In this study, 61 CCCH genes within C. bungei were identified and characterized. Phylogenetic assessment divided these genes into 9 subfamilies, with 55 members mapped across 16 chromosomes. The analysis of gene structures and protein motifs indicated that members within the same subfamily shared similar exon/intron distribution and motif patterns, supporting the phylogenetic classification. Collinearity analysis suggested that segmental duplications have played a significant role in the expansion of the C. bungei CCCH gene family. Notably, RNA sequencing analysis under 4 °C cold stress conditions identified CbuC3H24 and CbuC3H58 as exhibiting the most significant responses, highlighting their importance within the CCCH zinc finger family in response to cold stress. The findings of this study lay a theoretical foundation for further exploring the mechanisms of cold tolerance in C. bungei, providing crucial insights for its cultivation in cold regions.

Effects of nitrate- and ammonium- nitrogen on anatomical and physiological responses of Catalpa bungei under full and partial root-zone drought.

Catalpa bungei is a precious timber species distributed in North China where drought often occurs. To clarify adaptive responses of C. bungei to partial- and full- root-zone drought under the influence of nitrogen forms, a two-factor experiment was conducted in which well-watered (WW), partial root-zone drought in horizontal direction (H-PRD) and in vertical direction (V-PRD), and full root-zone drought (FRD) were combined with nitrate-nitrogen (NN) and ammonium-nitrogen (AN) treatments. C. bungei responded to FRD by sharply closing stomata, decreasing gas exchange rate and increasing leaf instantaneous water use efficiency (WUEi). Under FRD condition, the growth of seedlings was severely inhibited and the effect of N forms was covered up by the drastic drought effect. In comparison, stomata conductance and gas exchanges were moderately inhibited by PRDs. WUEi in V-PRD treatment was superior to H-PRD due to the active stomata regulation resulting from a higher ABA level and active transcription of genes in abscisic acid (ABA) signaling pathway under V-PRD. Under both PRDs and FRD, nitrate benefited antioxidant defense, stomata regulation and leaf WUEi. Under V-PRD, WUEi in nitrate treatment was superior to that in ammonium treatment due to active stomata regulation by signaling network of nitric oxide (NO), Ca2+ and ABA. Under FRD, WUEi was higher in nitrate treatment due to the favoring photosynthetic efficiency resulting from active NO signal and antioxidant defense. The interactive effect of water and N forms was significant on wood xylem development. Superoxide dismutase (SOD) and catalase (CAT) largely contributes to stress tolerance and xylem development.

Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpabungei.

As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. Although the SPL gene family has been identified in diverse plant species, there have been no genome-wide identification or systematic study reports on the SPL gene family in Catalpa bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome. According to the phylogenetic relationship, they can be divided into eight groups, and the genes in the same group have a similar gene structure and conserved motifs. Synteny analysis showed that fragment duplication played an important role in the expansion of the CbuSPL gene family. At the same time, CbuSPL genes have cis-acting elements and functions related to light response, hormone response, growth and development, and stress response. Tissue-specific expression and developmental period-specific expression analysis showed that CbuSPL may be involved in flowering initiation and development, flowering transition, and leaf development. In addition, the ectopic expression of CbuSPL4 in Arabidopsis confirmed that it can promote early flowering and induce the expression of related flowering genes. These systematic research results will lay a foundation for further study on the functional analysis of SPL genes in C. bungei.

Genome-wide analysis of lncRNA and mRNA expression and endogenous hormone regulation during tension wood formation in Catalpa bungei.

BACKGROUND: Phytohormones are the key factors regulating vascular development in plants, and they are also involved in tension wood (TW) formation. Although the theory of hormone distribution in TW formation is widely supported, the effects of endogenous hormones on TW formation have not yet been assessed. In this study, TW formation was induced in Catalpa bungei by artificial bending. The phytohormone content of TW, opposite wood (OW) and normal wood (NW) was determined using liquid chromatography-mass spectrometry (LC-MS), and transcriptome sequencing was performed. The hormone content and related gene expression data were comprehensively analyzed. RESULTS: The results of analyses of the plant hormone contents indicated significantly higher levels of cis-zeatin (cZ), indoleacetic acid (IAA) and abscisic acid (ABA) in TW than in OW. Genes involved in the IAA and ABA synthesis pathways, such as ALDH (evm. MODEL: group5.1511) and UGT (evm. MODEL: scaffold36.20), were significantly upregulated in TW. and the expression levels of ARF (evm. MODEL: group5.1332), A-ARR (evm. MODEL: group0.1600), and TCH4 (evm. MODEL: group2.745), which participate in IAA, cZ and Brassinolide (BR) signal transduction, were significantly increased in TW. In particular, ARF expression may be regulated by long noncoding RNAs (lncRNAs) and the HD-ZIP transcription factor ATHB-15. CONCLUSIONS: We constructed a multiple hormone-mediated network of C. bungei TW formation based on hormone levels and transcriptional expression profiles were identified during TW formation.

Multi-omics sequencing provides insight into floral transition in Catalpa bungei. C.A. Mey.

BACKGROUND: Floral transition plays an important role in development, and proper time is necessary to improve the value of valuable ornamental trees. The molecular mechanisms of floral transition remain unknown in perennial woody plants. "Bairihua" is a type of C. bungei that can undergo floral transition in the first planting year. RESULTS: Here, we combined short-read next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to provide a more complete view of transcriptome regulation during floral transition in C. bungei. The circadian rhythm-plant pathway may be the critical pathway during floral transition in early flowering (EF) C. bungei, according to horizontal and vertical analysis in EF and normal flowering (NF) C. bungei. SBP and MIKC-MADS-box were seemingly involved in EF during floral transition. A total of 61 hub genes were associated with floral transition in the MEturquoise model with Weighted Gene Co-expression Network Analysis (WGCNA). The results reveal that ten hub genes had a close connection with the GASA homologue gene (Cbu.gene.18280), and the ten co-expressed genes belong to five flowering-related pathways. Furthermore, our study provides new insights into the complexity and regulation of alternative splicing (AS). The ratio or number of isoforms of some floral transition-related genes is different in different periods or in different sub-genomes. CONCLUSIONS: Our results will be a useful reference for the study of floral transition in other perennial woody plants. Further molecular investigations are needed to verify our sequencing data.

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei.

BACKGROUND: "Bairihua", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. RESULTS: SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in "Bairihua" and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in "Bairihua". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. CONCLUSIONS: Our results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

Transcriptomics and Proteomics Reveal the Cellulose and Pectin Metabolic Processes in the Tension Wood (Non-G-Layer) of Catalpa bungei.

: Catalpa bungei is an economically important tree with high-quality wood and highly valuable to the study of wood formation. In this work, the xylem microstructure of C. bungei tension wood (TW) was observed, and we performed transcriptomics, proteomics and Raman spectroscopy of TW, opposite wood (OW) and normal wood (NW). The results showed that there was no obvious gelatinous layer (G-layer) in the TW of C. bungei and that the secondary wall deposition in the TW was reduced compared with that in the OW and NW. We found that most of the differentially expressed mRNAs and proteins were involved in carbohydrate polysaccharide synthesis. Raman spectroscopy results indicated that the cellulose and pectin content and pectin methylation in the TW were lower than those in the OW and NW, and many genes and proteins involved in the metabolic pathways of cellulose and pectin, such as galacturonosyltransferase (GAUT), polygalacturonase (PG), endoglucanase (CLE) and ß-glucosidase (BGLU) genes, were significantly upregulated in TW. In addition, we found that the MYB2 transcription factor may regulate the pectin degradation genes PG1 and PG3, and ARF, ERF, SBP and MYB1 may be the key transcription factors regulating the synthesis and decomposition of cellulose. In contrast to previous studies on TW with a G-layer, our results revealed a change in metabolism in TW without a G-layer, and we inferred that the change in the pectin type, esterification and cellulose characteristics in the TW of C. bungei may contribute to high tensile stress. These results will enrich the understanding of the mechanism of TW formation.

Construction of a high-density genetic map and QTL mapping of leaf traits and plant growth in an interspecific F1 population of Catalpa bungei × Catalpa duclouxii Dode.

BACKGROUND: Catalpa bungei is an important tree species used for timber in China and widely cultivated for economic and ornamental purposes. A high-density linkage map of C. bungei would be an efficient tool not only for identifying key quantitative trait loci (QTLs) that affect important traits, such as plant growth and leaf traits, but also for other genetic studies. RESULTS: Restriction site-associated DNA sequencing (RAD-seq) was used to identify molecular markers and construct a genetic map. Approximately 280.77 Gb of clean data were obtained after sequencing, and in total, 25,614,295 single nucleotide polymorphisms (SNPs) and 2,871,647 insertions-deletions (InDels) were initially identified in the genomes of 200 individuals of a C. bungei (7080) × Catalpa duclouxii (16-PJ-3) F1 population and their parents. Finally, 9072 SNP and 521 InDel markers that satisfied the requirements for constructing a genetic map were obtained. The integrated genetic map contained 9593 pleomorphic markers in 20 linkage groups and spanned 3151.63 cM, with an average distance between adjacent markers of 0.32 cM. Twenty QTLs for seven leaf traits and 13 QTLs for plant height at five successive time points were identified using our genetic map by inclusive composite interval mapping (ICIM). Q16-60 was identified as a QTL for five leaf traits, and three significant QTLs (Q9-1, Q18-66 and Q18-73) associated with plant growth were detected at least twice. Genome annotation suggested that a cyclin gene participates in leaf trait development, while the growth of C. bungei may be influenced by CDC48C and genes associated with phytohormone synthesis. CONCLUSIONS: This is the first genetic map constructed in C. bungei and will be a useful tool for further genetic study, molecular marker-assisted breeding and genome assembly.

Genome-wide analysis of long non-coding RNAs in Catalpa bungei and their potential function in floral transition using high-throughput sequencing.

BACKGROUND: Long non-coding RNAs (lncRNAs) have crucial roles in various biological regulatory processes. However, the study of lncRNAs is limited in woody plants. Catalpa bungei is a valuable ornamental tree with a long cultivation history in China, and a deeper understanding of the floral transition mechanism in C. bungei would be interesting from both economic and scientific perspectives. RESULTS: In this study, we categorized C. bungei buds from early flowering (EF) and normal flowering (NF) varieties into three consecutive developmental stages. These buds were used to systematically study lncRNAs during floral transition using high-throughput sequencing to identify molecular regulatory networks. Quantitative real-time PCR was performed to study RNA expression changes in different stages. In total, 12,532 lncRNAs and 26,936 messenger RNAs (mRNAs) were detected. Moreover, 680 differentially expressed genes and 817 differentially expressed lncRNAs were detected during the initiation of floral transition. The results highlight the mRNAs and lncRNAs that may be involved in floral transition, as well as the many lncRNAs serving as microRNA precursors. We predicted the functions of lncRNAs by analysing the relationships between lncRNAs and mRNAs. Seven lncRNA-mRNA interaction pairs may participate in floral transition. CONCLUSIONS: This study is the first to identify lncRNAs and their potential functions in floral transition, providing a starting point for detailed determination of the functions of lncRNAs in C. bungei.