Differential Analysis of Nutrient Content and Metabolism of Camellia oleifera at Different Growing Periods

Summary

This article presents a protocol to provide the first report on the nutrient content and related metabolic pathways of C. oleifera leaves at three growth and development stages, and to provide a basis for understanding the molecular mechanism of leaf senescence.

Abstract

In order to indicate the variations in nutrient content, gene expression associated with nitrogen metabolism, and starch and sucrose metabolism in C. oleifera at different growing periods, leaves from the C. oleifera 'Dabieshan 1' cultivar were collected at the juvenile (2-year-old C. oleifera), early fruiting stage(6-year-old C. oleifera), and peak fruiting stages(10-year-old C. oleifera), which were selected as experimental materials. Determination of nutrient content and analysis of transcriptome sequencing were conducted on leaf samples in the early flowering stage, systematically studying the content of nutrients and related metabolic pathways of C. oleifera during different growing periods. The results distinguished 9 homologous genes related to nitrogen metabolism and 24 related to starch and sucrose metabolism, respectively, after subjecting the C. oleifera leaves collected during both the juvenile and the peak fruiting stage to comparison in the KEGG database. With increasing tree age, changes in the nitrogen metabolism and the starch and sucrose metabolic pathways in C. oleiferaleaves. C. oleifera would enhance both the phenylalanine ammonia-lyase pathway and sucrose biosynthesis. These ultimately led to increased levels of soluble proteins and soluble sugars in C. oleifera leaves. This study provides new insights into the mechanisms of changes in nitrogen metabolism, starch and sucrose metabolism, and nutrient content during the transformation of C. oleifera from vegetative growth to reproductive growth. It offers theoretical references for understanding its fruiting period and applying plant foliar fertilizers to promote flowering and fruiting.

Introduction

During its juvenile stage, C. oleifera typically possesses an undivided main stem and primarily undergoes vegetative growth, lacking the capability for reproductive processes like flower bud differentiation and flowering¹. When grafting seedlings of C. oleifera, it is common practice to use spring shoots entering the adult stage as scions, as they have the ability to induce flowering^2,3,4. However, during the early planting stage, C. oleifera in its juvenile stage may struggle to form flower buds and undergo flower bud differentiation due to vigorous vegetative growth, as environmental conditions may limit flower bud formation. Nonetheless, as C. oleifera progresses through its juvenile stage to its primary fruiting period and eventually reaches its fruiting peak, it gradually develops the ability to undergo normal flower bud formation and flowering. C. Oleifera has the characteristic of a complex shape, simultaneous fruits and flowers⁵. In the initial fruiting stage, C. oleifera may produce a few flowers, but when it enters the peak fruiting stage, it produces a greater quantity of flowers. At the peak fruiting stage of C. oleifera, reproductive growth becomes predominant, accompanied by increased demands for light, temperature, water, and nutrients. It is evident that woody fruit trees, like C. oleifera, have a long vegetative growth period before flowering, limiting the possibility of shortening the cultivation cycle and realizing early high productivity⁶.

Nitrogen is super important for how plants grow and develop. Different types of plants have different amounts of nitrogen, and even within the same species, significant disparities in nitrogen content exist at different growing periods. As plants transition into the reproductive growth stage, nitrogen gradually accumulates in organs such as flowers, fruits, and seeds⁷. Nitrogen is essential for enzymes, proteins, specific hormones, nucleic acids, and other vital substances, thereby orchestrating metabolic activities⁸. The process of flower bud differentiation constitutes a key turning point, transitioning plants from their vegetative phase into the reproductive stage, and involves an intricate developmental process. The accumulation of nutrients serves as a fundamental basis for the differentiation of plant flower buds⁹. Current consensus holds that successful flower bud differentiation hinges on carbohydrate accumulation¹⁰. Throughout fruit tree flower bud differentiation, fluctuations in soluble sugar content directly or indirectly influence the process¹⁰. Investigations have shown a close correlation between carbohydrate levels in Prunus salicina leaves and flower bud differentiation, as the soluble sugar levels is higher in the physiological flower bud differentiation phase than the Floral bud morphological differentiation period, for P. salicina underwent a rapid depletion of carbohydrates during flower bud differentiation¹¹. Nevertheless, there are reports indicating that no significant fluctuation in soluble starch content is observed in Ficus carica during flower bud differentiation¹². Evergreen plants accrue starch within the ovary during the spring period of flower bud differentiation, while leaf-falling plants store starch during the bud dormancy period¹³. Based on the above information, we could conclude that there are differences in the trajectories of carbohydrate content changes among different species during flower bud differentiation. Ying et al.¹⁴ analyzed the patterns of expression of key enzyme genes implicated in basic meteorological pathways processes, such as nitrogen metabolism, and the metabolism of sucrose and starch, at two different stages of floral bud differentiation in Vernicia fordii. This analysis was combined with the dynamic changes in physiological and biochemical indicators of tung tree floral buds, providing a detailed elucidation of nutrient accumulation, utilization, transport, and distribution during tung tree floral bud differentiation^8,14. Research by Lai Chengchun et al. found that removing two leaves can modulate the expressed genes sucrose phosphate synthase (SPS), neutral invertase (NI), cell wall invertase (CWI), and α-amylase (AMY), thereby promoting saccharose synthesis and the subsequent accumulation of starch, providing the essential nutritional framework necessary for fruit maturation, shoot sprouting, and flowering^13,15.

However, research on changes in nutrient content, nitrogen metabolism, and starch and sucrose metabolism in C. oleifera at different stages was rarely reported. Most studies have focused on constructing single-nucleotide polymorphism (SNP) maps, developing SSR markers, and dissecting the physiological and molecular responses of C. oleifera under low or high temperature conditions^{16,17,18,19,20}. Therefore, in this study, the common C. oleifera cultivar "Dabieshan 1" was selected, and leaf samples of spring shoots' at different growing periods (juvenile stage, initial fruiting stage, and peak fruiting stage) of C. oleifera were collected for nutrient and transcriptome sequencing analysis. The aim was to identify differences in nutrient content, nitrogen metabolism, and the metabolism of sucrose and starch gene expression across the various phases throughout the processes of growth and development observed in C. oleifera leaves. Clarifying the nitrogen metabolism, sucrose and starch metabolism pathways, and their relationships with related nutrients can lay the foundation for studying the mechanism of C. oleifera floral bud differentiation and shed light on the molecular mechanisms underlying C. Oleifera flowering.

Protocol

1. Experimental materials

C. oleifera 'Dabieshan 1' leaves were collected at three key stages of development: the juvenile stage, at around 2 years of age; the subject progresses to an early fruiting phase at approximately 6 years old, eventually reaching its peak fruiting period when it is about 10 years old. All the C. oleifera plants at the above three developmental stages were planted using container-grown seedlings of the same specification of C. oleifera 'Dabieshan 1'. The physical and chemical properties of the soil in C. oleifera forest land were shown in Table 1.

2. Collection site overview

Experimental materials were collected from Heping Town, located in the heart of Anhui Province. Shucheng County lies within the province's central region, belonging to the peripheral area of the Dabie Mountains. The coordinates are 31°11 ′N, 116°47 ′E, with a mean elevation of 90 m. The soil is classified as yellow-brown soil. The region has a humid subtropical monsoon climatic pattern, which maintains an average annual temperature of 15.6 °C and typically sees around 1,100 mm of rainfall each year, enjoys approximately 1,969 h of sunshine each year, and has a frost-free period lasting 224 days.

3. Sample collection and pretreatment

Samples were selected during the pre-flowering period (late September). For each developmental stage, the C. oleifera plants with similar, consistent, and strong growth were selected. From each plant, the third leaf was removed from top to bottom, which was moderately growing spring shoots from the east, west, south, and north directions of the outer part of the C. oleifera crown. The samples were marked with serial letters and numbers: YAT1, YAS2, and YAT3 for C. oleifera leaves at the juvenile stage, the subject progresses to an early fruiting phase, and the peak fruiting period, respectively. These were collected and stored in liquid nitrogen. Five random sample trees were selected from each stage as one biological replicate, and three biological replicates were set up in all stages. The selected specimens were brought back the same day to the laboratory and preserved at -80 °C.

4. Physiological index determination

The soluble sugar content was determined by the anthrone colorimetric method²¹. Organic carbon content was determined by the potassium dichromate oxidation - ferrous sulfate titration method²². The soluble protein content was obtained by the Coomassie brilliant blue method²³. The total nitrogen concentration wasquantified by the Kjeldahl method²².

5. Isolation of complete RNA was conducted, followed by the construction of a complementary DNA (cDNA) library and subsequent sequencing

Total RNA was extracted from C. oleifera leaves at different stages using the plant RNA extraction kit. Total RNA concentration was determined by a full-spectrum ultraviolet-visible spectrophotometer. The integrity and clarity of RNA were determined by a biological analyzer. After constructing and validating the cDNA library, transcriptomic sequencing was conducted utilizing a sequence-by-sequencing high-throughput sequencing platform. The sequencing procedure produced individual reads, each spanning 100 base pairs^24,25,26. The sequence process alignment results underwent statistical analysis using the RSEM software²⁷. Differential expression genes (DEGs) at various developmental stages of C. oleifera were identified using the DESeq differential analysis software^28,29.

6. De novo assembly and UniGene annotation

SeqPrep http://github.com/jstjohn/SeqPrep) and Sickle (http://github.com/najoshi/sickle) were used to filter the raw data, including removing low-quality sequences and contaminated adapters. Then the clean reads obtained from each sample were assembled using Trinity software^30,31. All the assembled transcripts were subjected to BLASTX alignment against the Swiss-Prot, Clusters of orthologous groups for eukaryotic complete genomes, and KEGG databases in sequential order. A typical Cut-off E-value (E-value <10⁻⁵) was set to retrieve their function annotations. The KEGG was applied to analyze related metabolic pathways³².

7. Functional annotation of genes and differentially expressed genes analysis

Gene and isoform abundances were statistically analyzed using the RSEM software²⁷. A cut-off (P < 0.05, FDR < 0.01, |log2FC|>1) was set to identify differentially expressed genes (DEGs). The expected number of fragments per kb of transcript per million fragments mapped (FPKM) is currently the most widely used method for estimating gene expression levels²⁸. The FPKM values of all genes from the three different growth and developmental stages (YAT1, YAS2, and YAT3) were calculated. Hierarchical clustering analysis was conducted to examine the differential gene expression levels. Meanwhile, the KEGG pathway was prepared to identify DEGs significantly enriched in metabolic pathways at Bonferroni-corrected P ≤ 0.05 compared to the whole transcriptome using KOBAS²⁹.

8. Analytical methods

The data were presented using Excel to calculate mean values and standard deviations. Analysis of variance (ANOVA) was used for statistical analysis by SPSS software, and Duncan's multiple range test was used to assess significant differences in nutrient content among the three stages. Then, significant differences between groups were determined using post-hoc multiple comparisons by the Least Significant Difference (LSD) method. Graphs and charts were generated using Origin software to visualize experimental and analytical findings. No statistical significance was denoted by 'ns' (P > 0.05). Statistically significant differences were denoted by '*' (P < 0.05), '**' (P < 0.01), '***' (P < 0.001), and '****' (P < 0.0001), respectively.

Results

Comparison of nitrogenous compound content in C. oleifera leaves at different growing periods

Nitrogen was paramount in facilitating the growth and development of plants, exerting a profound influence on their reproductive processes. Consequently, comprehending the overall nitrogen content in plants holds considerable significance for research endeavors. The total nitrogen content in C. oleifera leaves showed a gradual reduc...

Discussion

Soluble sugars serve as essential energy sources and structural components in plants, fueling various physiological activities and biosynthetic processes¹⁰. Alterations in soluble sugar content directly or indirectly influence the process of transforming vegetative buds into flower buds in fruit trees¹⁰. Some research has found that the necessity of abundant soluble sugars was for fruit tree flower bud differentiation³³. Soluble proteins play a cruci...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bioanalyzer 2100	Agilent Technologies, Inc. (USA)	—	Used to evaluate RNA integrity and clarity
DESeq software	Source code	3.2	Used for differential expression analysis
EASYspin Plus kit (Total RNA isolation)	Aidlab Biotechnology Co., Ltd. (China)	RN 28 (Aidlab)	Used for total RNA extraction from C. oleifera leaves
HiSeq 2000 sequencing platform	Illumina (China)	—	Used for transcriptome sequencing, read length 100 bp
Kjeldahl method/Kjeltec 2300	Foss Tecator(Sweden)	—	For total nitrogen concentration(Bao 2007)
Method for soluble protein determination	Concentrated sulfuric acid	6104-58-1(aladdin)	Method developed by Chen Jianxun (Chen et al. 2015)
Method for soluble sugar determination	Anthrone	90-44-8 (Macklin)	Zhang Zhilang’s method (Zhang et al. 2003)
Potassium dichromate oxidation - ferrous sulfate titration method	Potassium dichromate Ferrous Sulfate	NA (aladdin) 7782-63-0(aladdin)	For organic carbon determination (Bao 2007)
RSEM software	Source code	1.2.3	Used for sequence alignment statistical analysis
Thermo Scientific NanoDrop 2000C spectrophotometer	ABI(USA)	—	Used to determine RNA concentration