Transcriptome Analysis Provides Insights into Korean Pine Tree Aging and Response to Shading

Transcriptome Analysis Provides Insights into Korean Pine Tree Aging and Response to Shading

1. Introduction

Pollution and climatic degradation have led to our focus on age-related changes in tree development and physiology because trees of different ages respond differently to the environment. For example, after heat stimulation, cambium cells in younger trees resume their activities earlier, showing strong sensitivity to temperature [1]. Moreover, elevated resistance to pathogens occurs with aging in apple trees and other flowering plants [2,3,4,5]. Sensitivity to the environmental cues that induce flowering increases with tree age, and this provides a core basis for the separation of the juvenile vegetative phase from the adult vegetative phase [6,7]. These studies indicate that the developmental and physiological responses of trees change with age. However, how age controls the changes in these responses in trees is unknown.
Shading affects light cues, influencing plant growth and development, and this has been well studied in crops. For example, the leaf angle affects a plant’s photosynthetic efficiency and yield [8], and the molecular mechanism of this factor has been widely reported in rice and maize, providing a possibility for the quantitative control of leaf angles at different canopy levels [9]. Other methods like density control, dwarfing rootstock [10], and pruning [11], have been studied in fruit tree management and used to manipulate shading to improve yield. Shading also affects plant metabolism. For example, shading decreases the epicatechin, epicatechin gallate, and epigallocatechin levels, as well as increasing the caffeine levels in tea plants [12]; moreover, a higher vitamin C content has been found in blueberry fruit subjected to shading treatment [13].
In forest tree management, shading is also used to control tree growth. Korean pine (Pinus koraiensis, Sieb. et Zucc.) is a valuable crop and timber tree, and its pine nuts are a popular snack and raw material for oil production. Therefore, Korean pine is the most popular tree species for farmers in Northeast China; however, it takes about 14 years for a tree grown from a seed to bear fruit, resulting in slow returns. Furthermore, adult Korean pines are photophilous, but juvenile trees are resistant to shade, and proper shading does not suppress the growth of young Korean pines [14]. This habit is used to solve the problem of the slow artificial regeneration of Korean pine forests, known as “afforestation under canopy” (Figure 1). Usually, 4-year-old Korean pine saplings that are more than 35 centimeters tall are selected and planted with two-meter spacing under 30–40-year-old larch trees, which were planted with five-meter spacing.

We hypothesized that gene expression changes during tree aging, and age and shading effects on the development and physiology of Korean pine trees, can be detected at the transcriptional level. To prove this hypothesis, we investigated the transcriptomes of Korean pines of different ages and the effects of shading on them, providing a theoretical basis for Korean pine management.

2. Materials and Methods

2.1. Plant Material

Tree development and physiology vary at different heights. The apical stem, to some extent, is considered to represent the physiological age of a tree developed from a seed. To weaken position effects, the uppermost newly produced main stems were collected from 5-, 7-, 10-, 14-, and 17-year-old active Korean pine trees. Active 14- and 17-year-old Korean pine trees that were planted under the canopy were also sampled to study the effect of shading on the development and physiological responses of Korean pine trees. The trees under study were growing in different stands in the Dabiangou seed orchard (42°02′ N, 125°07′ E), Liaoning Province, in Northeast China (Table 1), and all trees were grown from seeds.
Sample collection was performed on the morning of 31 July 2021. Secondary growth mediated by cambium and lignification occurred actively in these sampled stems. When sampling, we found that some 14- and 17-year-old trees bore cones and these were collected separately (Table 1). Each sample had three replicates. After the removal of branches and needles, a 15-cm segment (from the top) of the main stem of each tree was cut, immediately frozen in dry ice, and then stored at −80 °C until RNA extraction.

2.2. CDNA Library Preparation and Transcriptome Sequencing

Total RNA extraction from each tree was performed using a plant RNA purification reagent, as per the manufacturer’s instructions (Invitrogen, San Diego, CA, USA). RNA was quantified using a Qubit RNA kit (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Five micrograms of total RNA from each tree was used to generate an RNA library for Illumina paired-end sequencing. In total, 27 libraries were constructed.

After isolation from 5 μg of total RNA, the mRNA was purified using mRNA capture beads and then broken into 300–500 bp short fragments using magnetic beads. Subsequently, these fragments were employed as templates for the synthesis of first-strand cDNA, using a random hexamer primer. Then, second-strand cDNA was synthesized and cleaned up to prepare for end repair and the addition of poly A. Thereafter, these strands were connected to sequencing adapters and the desired cDNA was isolated using Hieff NGS® DNA Selection Beads (YEASEN, Shanghai, China). To enrich the desired cDNA, PCR amplification was performed. PCR products were tested on 2% agarose gel. Finally, the libraries were sequenced using the Illumina HiSeqTM 2500 platform (Illumina, CA, USA).

2.3. Transcriptome Assembly and Annotation

To identify the Korean pine protein-coding genes, blastx was used to search our assembled sequences against the blastx_swissprot, protein, blastp_swissport, Pfam, SignalP, eggnog, blast2GO, Pfam2GO, and Kegg databases with an E value < 1 × 10−5. Gene Ontology (GO) terms associated with the top hits in the nine protein databases were assigned to the annotated transcripts. After sequence clustering with Trinity software, unigenes were obtained and used for further analysis.

2.4. Identification of Differentially Expressed Unigenes (DEUs)

To identify the DEUs, 27 sets of sequencing reads were mapped pairwise to the assembled reference transcripts, and 36 comparisons were produced. Then, the normalized expression value was calculated as the fragments per kilobase of transcript per million mapped reads (FPKM). A pairwise comparison between two samples was performed to identify the DEUs with a false discovery rate (FDR)-corrected p value cutoff of 0.05 and a minimum expression fold change of 2.

2.5. Statistical Analyses and Graphics

Multiple comparisons were used to obtain the significance levels through the ANOVA Duncan method using SPSS version 26. The relationships between the gene expression patterns and age were analyzed with simple linear regression using GraphPad Prism version 9. Heatmaps were used to show the gene expression pattern using TBtools v. 2.04 [15], and log and row scales were used to display the changes in patterns between groups.

4. Discussion

While this study focused on age and shading, other environmental factors, such as soil quality, water availability, and pest presence, may also significantly impact tree growth and physiology. In this work, the sampled trees grew closely, so other environmental factors had a lower impact on their growth and physiology. In the future, the influences of other environmental factors can be studied further. In conclusion, our findings offer valuable insights into how age and shading affect the development and physiological traits of Korean pine trees, providing a foundation for further studies and practical forest management strategies.

4.1. Transcriptomic Analysis Reveals the Age-Related Developmental and Physiological Changes in Korean Pine Trees at the Transcriptional Level

Traits like photosynthesis [22], the stress response [5], and reproduction [6] change dynamically during tree aging, and studying their underlying mechanisms helps us to understand tree adaptation and utilize this in forest management. In this study, based on transcriptomic analysis, DEUs associated with stress, light, temperature, and reproduction were identified from trees of different ages after pairwise comparisons. Our findings demonstrate that these traits change in Korean pine trees with age, it was possible to capture these changes via our transcriptomic analysis. More importantly, 10 DEUs associated with the stress response showed age-related expression patterns (Figure 2), indicating that stress responses might be mediated by these DEUs and may change as Korean pines age. Some DEUs are associated with the temperature response (Figure 3) and sexual reproduction (Figure 4), revealing that tree age coordinates these traits. Furthermore, studying the regulation of these DEUs will help us to understand how age controls these developmental and physiological traits.

4.2. Shading Affects the Expression of Physiological Response-Related Genes in Korean Pine

Light is a major environmental cue for plants [23], and a reduction in light intensity restrains plant growth in most species [24,25]. Plants respond to light in several ways and interact with the neighboring plants to avoid shading [12,26]. However, shading also protects seedlings from photo-oxidative damage in the early stage [27,28], and can even increase biomass and positively influence plant growth [29]. Altogether, the effect of shading on plant growth has two sides, and studying it is important for effective planting density adjustment, forest space allocation, and understory economic management [30]. Here, we provide molecular evidence that shading changes the physiological responses in Korean pine trees. For example, we identified 10 DEUs that were involved in the stress response, and their expression levels were changed by shading (Figure 2). Shading also decreased the expression levels of 15 heat shock protein genes (Figure 3). These results demonstrate that shading changes the expression of genes associated with stress, light, and temperature responses in Korean pine trees.

4.3. The Expression Level of PkDAL1 Is Age-Dependent, Independent of the Reproductive State or Growth Conditions of Korean Pine Trees

PkDAL1 showed an increased expression pattern during Korean pine aging (Figure 4). In a previous work, a transcript (c77350.graph_c2) annotated as Picea abies DAL1 was identified as being differentially expressed between 2- and 5-, 10-, 15-, or 30-year-old Korean pine trees [31]. The age-dependent expression patterns of DAL1 homologs have also been characterized in P. abies [32], P. tabuliformis [33], and Larix kaempferi [34,35,36]. Here, we found that there was a positive correlation between the expression pattern of PkDAL1 and tree age (Figure 4b), and its expression level was almost the same in trees of the same age but with different growth conditions or reproductive states (Figure 4a). Taken together, these results indicate that the expression level of PkDAL1 is age dependent and independent of reproductive state or growth conditions. These findings are consistent with our previous study because there was no difference in L. kaempferi DAL1 (LaDAL1) transcript levels between 13-year-old larch trees in different reproductive states [36], further confirming its role as an age marker in conifers.
Notably, overexpression of DAL1 homologs promotes flowering in Arabidopsis thaliana [32,33,37,38]. In addition, LaDAL1 accelerates the other life-cycle events in Arabidopsis, as well as flowering, by promoting the transition of meristem fate [38]. These studies indicate that the conserved function of DAL1 homologs in the control of life-cycle progression may also exist in Korean pine trees.

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