Comparative Transcriptomic and Physiological Analyses Reveal Key Factors for Interstocks to Improve Grafted Seedling Growth in Tangor

Abstract

Interstock is an important agronomic technique for regulating plant growth and fruit quality, and overcoming the incompatibility between rootstocks and scions; however, the underlying mechanisms remain largely unknown. In this study, the effects and regulatory mechanisms of tangor grafting, with and without interstocks, on the growth and development of scions were analyzed by combining morphology, physiology, anatomy and transcriptomics. Morphological and physiological analyses showed that interstocks ('Aiyuan 38' and 'Daya') significantly improved the growth of seedlings, effectively enhanced the foliar accumulation of chlorophyll and carotenoids, and increased the thickness of leaf tissues. Using 'Aiyuan 38' as the interstock, photosynthetic efficiency and starch content of citrus seedlings improved. Transcriptomics showed that genes related to photosynthesis and photosynthetic antenna proteins were upregulated in interstock-treated seedlings, with significant upregulation of photosystem PSI- and PSII-related genes. In addition, multiple key genes may be involved in plant hormone signaling, starch and sucrose metabolism, and transcriptional regulation. Taken together, these findings provide novel insights into the role of interstocks in regulating and contributing to the growth and development of grafted seedlings, and will further define and deploy candidate genes to explore the mechanisms of rootstock-interstock-scion interactions.

Keywords: Mingrijian; grafting; interstock; rootstock; tangor; transcriptome.

Figure 1

The effect of interstocks on the growth responses of ‘Mingrijian’ citrus. (A) Grafting diagram of seedlings used in this experiment. (B) Effects of interstocks on the growth morphology of ‘Mingrijian’ after 210 days. (C) The length of new shoots, diameter of new shoots, and plant height of grafted seedlings under different interstocks. Abscissa coordinates represent different days after grafting. Longitudinal coordinates represent the increment of indexes. Data were the mean ± standard error of three replicates. Error bars indicate the standard error of total growth, and different letters indicate significant differences in total growth, p < 0.05. (D) Leaf cross-cutting structure (×200) of ‘Mingrijian’ after 210 days of grafting onto different interstocks.

Figure 2

Effect of different interstocks on photosynthetic related physiological indicators of grafted ‘Mingrijian’ seedlings leaves. (A) Microscopy of stoma from plants after 210 days, (B) total chlorophyll, (C) Pn, (D) starch content and (E) soluble sugar content. Data were the mean ± standard error of three replicates. Error bars indicate the standard error, and different letters indicate significant differences, p < 0.05.

Figure 3

The phytohormone levels in leaves under different interstock treatments. (A) IAA: indole-3-acetic acid, (B) ABA: abscisic acid, (C) CTK: cytokinin, (D) GA₃: gibberellin A₃. Data were the mean ± standard error of three replicates. Error bars indicate the standard error, and different letters indicate significant differences, p < 0.05.

Figure 4

Overview of transcriptomics changes of different comparisons affected by interstocks. (A) DEGs in three comparisons. (B) Venn diagram of DEG numbers in different comparison groups. The number on the left of the red arrow indicates upregulated gene number, and the number on the left of the green arrow indicates downregulated gene number. (C) KEGG analysis (top 5) in three comparisons.

Figure 5

Heat map shows the differentially expressed genes in the bHLHs, C2H2s and MYBs transcription factor families. Heat map of DEGs was drawn using the log2 fold-change value obtained from the pairwise comparison of samples. Red and blue indicate upregulation and downregulation, respectively.

Figure 6

Interstocks induced changes in the expression profiles of photosynthesis-related genes in three comparisons. (A) Photosynthesis pathway genes, (B) photosynthesis–antenna proteins pathway genes, and (C) heat map of photosynthesis-related gene expression. The rectangles behind the gene, which were tagged with red, green, white and yellow color, represent the upregulated DEGs, downregulated DEGs, unchanged DEGs, and the DEGs with inconsistent expression trends, respectively.

Figure 7

Interstocks induced changes in the expression profiles of hormone signaling pathway genes in three comparisons. The rectangles behind the gene, which were tagged with red, green, white and yellow color, represent the upregulated DEGs, downregulated DEGs, unchanged DEGs and the DEGs with inconsistent expression trends, respectively.

Figure 8

Interstocks induced changes in the expression profiles of the key starch and sugar metabolism. The rectangles behind the gene, which were tagged with red, green, white and yellow color, represent the upregulated DEGs, downregulated DEGs, unchanged DEGs and the DEGs with inconsistent expression trends, respectively.