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 differ....

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 Tab....

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