Comparative Analysis Based on Physiological and Transcriptomic Data between Juvenile and Adult Tree Peony (Paeonia delavayi)

Question

1. What are the physiological and transcriptomic differences between the leaves of juvenile and adult plants in P. delavayi?

2. What are the physiological differences between juvenile and adult P. delavayi leaves?

3. What is the quality of sequencing data obtained for juvenile and adult plants?

4. What is the total clean data obtained after sequencing cDNA libraries?

Accepted Answer

Previous studies have mainly focused on P. ostii or P. suffruticosa, the congeneric species to P. delavayi. Therefore, comparative physiological and transcriptomic analyses in P. delavayi leaves between juvenile and adult plants have not yet been performed. This study aims to reveal the physiological and transcriptomic differences between the leaves of juvenile and adult plants in P. delavayi. The results will provide a new perspective and help to reveal the possible regulatory mechanism of the juvenile-to-adult transition. In previous research, it was found that different plants have different change patterns with respect to the contents of carbohydrates and endogenous hormones in the juvenile and adult leaves. RNA-Seq was used to screen some key factors related to the juvenile-to-adult transition and flowering, such as SPL. Significant progress has been made on SPL in both model plants and some woody plants. The most typical function of SPL is promoting the juvenile-to-adult transition and flowering through the miR156/SPL module. In P. delavayi, 16 PsSPL genes were identified, and 6 of them were proved to be closely related to age via a qRT-PCR analysis. The results of this study will contribute to a better understanding of the juvenile-to-adult transition and flowering process in P. delavayi.

Accepted Answer

The physiological differences between juvenile and adult P. delavayi leaves include changes in starch and soluble sugar content, as well as hormone levels. In juvenile plants, the starch content remains unchanged, while the soluble sugar content is 4.91%. In adult plants, the soluble sugar content increases to 9.63%. The active GAs (GA1, GA3, and GA4), IAA, ABA, CTKs (cZ, tZ, cZR, tZR, iP, and iPR), ACC, and JA were determined in this study. However, GAs and CTKs-cZ, CTKs-tZ, CTKs-cZR, and CTKs-tZR were not detectable due to their trace levels or technology limit. Among the detectable hormones, the content of CTKs-iP in adult plants was significantly lower than in juvenile plants (1.14 ng*g-1 vs. 1.84 ng*g-1). The content of CTKs-iPR in adult plants was about one-third of that in juvenile plants, but there was no significant difference. The JA content in adult plants was significantly lower than in juvenile plants (0.12 ng*g-1 vs. 0.49 ng*g-1). Overall, these physiological differences provide insights into the juvenile-to-adult transition in P. delavayi leaves.

Accepted Answer

The sequencing data obtained for juvenile and adult plants is of high quality, with a percentage of Q30 bases at 92.34% and above. This indicates that the data is reliable and suitable for subsequent analysis. A total of 87.30Gb clean data was obtained after sequencing the cDNA libraries of the two groups, with each sample reaching 12.78 Gb. The high-quality sequencing data allows for accurate transcriptome sequencing assembly and annotation, ensuring the reliability of the results obtained.

Accepted Answer

A total of 87.30Gb clean data was obtained after sequencing the cDNA libraries of two groups of samples, juvenile (J) and adult (A). The clean data of each sample reached 12.78 Gb with a percentage of Q30 bases at 92.34% and above, indicating high-quality data suitable for subsequent analysis. This high-quality data allows for accurate transcriptome sequencing assembly and annotation, providing valuable insights into the genetic makeup of P. delavayi.

Accepted Answer

The unannotated DEGs related to tree peony's juvenile-to-adult phase transition and flowering have unknown functional enrichment pathways. However, the annotated DEGs mainly play roles in transportation and metabolism of carbohydrates and amino acids, translocation, defense response, and signal transduction, indicating enhanced transportation and metabolism during the adult phase. The five DEGs related to flowering, PdFUL, PdCOL, PdFPF1, PdLHY, and PdSPL10, showed up-regulated expression patterns and are enriched in the GO database domains of biological processes, cellular components, and molecular functions. The KEGG pathway enrichment analysis revealed that Circadian rhythm-plant and plant hormone signal transduction pathways are strongly associated with juvenile-to-adult transition and flowering.

Accepted Answer

SPL transcription factors play an important role in the age-dependent flowering pathway in P. delavayi. A total of 13 SPL genes were found in this study, including PdSPL1, PdSPL1A, PdSPL1B, PdSPL2, PdSPL7, PdSPL8-1, PdSPL8-2, PdSPL9, PdSPL10, PdSPL13, PdSPL13A, PdSPL14, and PdSPL16. Their expressions were all up-regulated in adult plants, with PdSPL10 showing a significant difference (5.64-fold higher expression in adult plants compared to juvenile plants). qRT-PCR verification results showed that the expression levels of 12 SPL genes in adult plants were higher than those in juvenile plants, except for PdSPL8-1. The fold difference of each SPL gene between juvenile and adult plants varied, with PdSPL8-2, PdSPL10, and PdSPL13 showing the highest differences (24.54, 6.66, and 6.24 times, respectively). PdSPL10 was the only differentially expressed SPL gene screened from the transcriptome data, suggesting its potential role during the juvenile-to-adult transition in P. delavayi.

Accepted Answer

Sugar signaling, possibly through the T6P pathway, plays a crucial role in mediating the juvenile-to-adult transition in P. delavayi. Carbohydrates, specifically starch and soluble sugar, are associated with this transition in plants. In P. delavayi, the sugar content in adult plants is significantly higher than that in juvenile plants. This increase in sugar content is consistent with previous studies in P. delavayi and R. chinensis. Soluble sugar not only provides energy for flowering but also acts as an endogenous signal to promote the juvenile-to-adult transition. The T6P pathway, which involves the conversion of UDP-glucose and glucose-6-phosphate into trehalose, is upregulated in adult P. delavayi plants. Trehalose functions as a signaling molecule that affects the juvenile-to-adult transition by repressing the miR156-SPL module. Therefore, the T6P pathway, along with the increase in sugar content, plays a significant role in the juvenile-to-adult transition in P. delavayi.

Accepted Answer

Hormones play a crucial role in regulating plant growth and development. In P. delavayi, the juvenile-to-adult transition involves the interaction of various hormones such as IAA, GA, CTK, ABA, JA, and ethylene. GA promotes flowering by degrading the transcriptional repressor DELLA protein and releasing SPL. High CTK levels are characteristic of juvenile tissues, while JA declines in advance of the transition. Although GAs were undetectable in P. delavayi leaves, IAA, ABA, and ACC showed no significant difference between two phases. The auxin pathway and gibberellin pathway were enriched in the enriched pathways, despite the undetectable GAs. The GA signaling pathway mediates the juvenile-to-adult transition through the miR156-SPL module, where DELLA protein interacts with SPL to inhibit flowering. SCL3 promotes GA signaling by antagonizing DELLA. The auxin pathway is also involved, with enriched auxin response factor 7 and SAUR32-like genes. The cross-talk between different hormone pathways suggests a complex mechanism that requires further exploration.

Accepted Answer

The circadian rhythm pathway, specifically the photoperiod pathway, has been found to be significantly enriched in the differentially expressed genes (DEGs) of P. delavayi. This finding aligns with previous studies on the flowering of related species such as P. suffruticosa and Pleioblastus pygmaeus. The circadian rhythm pathway is an endogenous timing mechanism that can sense light signals, including red and far-red light, through photosensitive cells of phytochrome and cryptochrome. The enrichment of DEGs in the circadian rhythm pathway suggests that it plays a crucial role in the transition from juvenile to adult and flowering in P. delavayi. Among the six enriched DEGs, except for the unannotated zf-B_box domain-containing protein (c70837), the homologous genes of the other five DEGs (PdCOL2, PdCDF3, PdLHY, PdRVE8, and PdRVE1) have been reported in other plants to be involved in growth and the flowering process. This indicates that the circadian rhythm pathway likely mediates the juvenile-to-adult process in P. delavayi.

Accepted Answer

SPL10 positively regulates the transition from juvenile to adult and flowering in P. delavayi. With juvenile-to-adult transition and flowering, PdSPL10 represses miR156 up-regulation, positively regulates downstream flowering-related genes (specifically PdFUL in this study), and miR172, promoting the juvenile-to-adult transition and flowering. In the genus Paeonia, PdSPL9 expression in P. delavayi buds increases with plant age, leading to shorter juvenile periods and earlier flowering time. Further experimental validation is needed to understand the function of PdSPL10.

Accepted Answer

In the hypothetical transcriptional regulatory network of the juvenile-to-adult transition in P. delavayi, 14 DEGs are associated with different pathways. These include 6 genes in the circadian rhythm pathway (PdCOL2, PdCDF3, PdLHY, PdRVE8, PdRVE1, and zf-B_box domain-containing protein), 3 genes in the plant hormone signal transduction pathway (PdARF19, PdSAUR32, and PdSCL3), 1 gene in the age pathway (PdSPL10), 1 gene in the downstream of the age pathway (PdFUL), and 3 genes in the sugar metabolism pathway (PdbGLU, PdTPS, and PdTPP). These genes are valuable for regulating the juvenile-to-adult transition in P. delavayi and can potentially facilitate breeding work in tree peony.

Accepted Answer

In the study, 30 juvenile and 30 adult plants of P. delavayi were used as experimental materials. These plants were grown at the tree peony planting base of the Institute of Vegetable and Flower, Chinese Academy of Agricultural Sciences (Yanqing Farm, Beijing). Fresh leaves were sampled during the flowering period in May 2021. Each phase of the samples contained three biological replicates, and a total of six samples were acquired. After quick freezing in liquid nitrogen, these samples were stored in a refrigerator at -80°C for further analysis.

Accepted Answer

Starch and soluble sugar contents were determined using the anthrone colorimetric method, as mentioned in references [64, 65]. This method involves the reaction of starch and soluble sugars with anthrone reagent, resulting in a color change that can be measured using a spectrophotometer. The absorbance values obtained are then used to calculate the concentration of starch and soluble sugars in the sample. This method is widely used in plant biochemistry research due to its simplicity and reliability.

Accepted Answer

In the Discovery of DEGs and Pathways section, DEGs are identified by first obtaining unigene sequences and expressing their expression levels as FPKM. Then, DESeq2 is performed to obtain DEGs between two groups of samples, using a false discovery rate (FDR) < 0.05 and fold change (FC) >= 2 as the difference screening criteria. The flowering-related DEGs are further identified based on functional annotation results. An enrichment analysis is performed using clusterProfiler, with GO and KEGG annotations based on InterProScan and KOBAS, and enrichment significance is calculated using a hypergeometric test.

Accepted Answer

SPL gene expressions were analyzed based on FPKM values. Real-time quantitative PCR (qRT-PCR) was performed using gene-specific primers to confirm the expression of these SPL genes. Actin was selected as the reference gene. Three biological replicates were mixed to extract RNA, and cDNA was obtained using the Vazyme Reverse Transcription Kit. The qRT-PCR reactions were performed with a Bio-Rad CFX96 real-time PCR instrument, following a specific procedure. The relative expression levels of the SPL genes were calculated using the 2-CT method, and all data were expressed as mean +- standard error (SE). Statistical analysis was conducted using the Student's t-test.

Accepted Answer

The soluble sugar content in the leaves of adult plants was significantly increased compared to juvenile plants, while the starch content remained unchanged. The contents of CTKs-iP and JA were significantly decreased, whereas the contents of IAA, ABA, CTKs-iPR, and ACC (ethylene precursor) remained unchanged. Circadian rhythm, plant hormone signal transduction, and sugar metabolism were closely related to the juvenile-to-adult transition in P. delavayi. The expression of the PdSPL genes was up-regulated in adult plants compared to juvenile plants, with PdSPL10 being differentially expressed and potentially playing a positive regulatory role in the juvenile-to-adult transition in P. delavayi. A hypothetical transcriptional regulatory network including 14 DEGs for the juvenile-to-adult transition in P. delavayi was constructed.

Comparative Analysis Based on Physiological and Transcriptomic Data between Juvenile and Adult Tree Peony (Paeonia delavayi)

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Most frequently asked questions

1. What are the physiological and transcriptomic differences between the leaves of juvenile and adult plants in P. delavayi?

2. What are the physiological differences between juvenile and adult P. delavayi leaves?

3. What is the quality of sequencing data obtained for juvenile and adult plants?

4. What is the total clean data obtained after sequencing cDNA libraries?

5. What are the functional enrichment pathways of the unannotated DEGs related to tree peony's juvenile-to-adult phase transition and flowering?

6. What is the role of SPL transcription factors in the age-dependent flowering pathway and how do their expressions differ between juvenile and adult plants in P. delavayi?

7. How does sugar signaling mediate juvenile-to-adult transition in P. delavayi?

8. How do hormones mediate juvenile-to-adult transition in P. delavayi?

9. What role does the circadian rhythm pathway play in P. delavayi's transition from juvenile to adult and flowering?

10. How does SPL10 regulate juvenile-to-adult transition in P. delavayi?

11. What pathways are associated with DEGs in P. delavayi's juvenile-to-adult transition?

12. What plant materials were used in the study?

13. How were starch and soluble sugar contents determined?

14. How are DEGs identified in the Discovery of DEGs and Pathways section?

15. How were SPL gene expressions analyzed in the transcriptome?

16. How are sugar metabolism and plant hormone signal transduction related to juvenile-to-adult transition in P. delavayi?

References

Analyzing real-time PCR data by the comparative C(T) method.

RNA-Seq: a revolutionary tool for transcriptomics

InterProScan 5: genome-scale protein function classification

KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases

Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3.

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