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. 2022 Jan 19:9:uhab072.
doi: 10.1093/hr/uhab072. Online ahead of print.

Comparative transcriptomic analysis on compatible/incompatible grafts in citrus

Wen He  1   2 , Rui Xie  1   2 , Yan Wang  1   2 , Qing Chen  1 , Hao Wang  1   2 , Shaofeng Yang  1   2 , Ya Luo  1 , Yong Zhang  1 , Haoru Tang  1   2 , Frederick G Gmitter  3 , Xiaorong Wang  1   2
Affiliations

Comparative transcriptomic analysis on compatible/incompatible grafts in citrus

Wen He et al. Hortic Res. .

Abstract

Grafting is a useful cultivation technology to resist abiotic and biotic stresses and is an integral part of citrus production. However, some widely utilized rootstocks may still exhibit graft incompatibility in the orchard. "Hongmian miyou" (Citrus maxima (Burm.) Merrill) is mutated from "Guanxi miyou", but these two scions showed different compatibility with available Poncirus trifoliata rootstock. Foliage etiolation is an observed symptom of graft incompatibility, but its mechanism remains poorly understood. This study is the first to investigate the morphological, physiological, and anatomical differences between the compatible/incompatible grafts, and perform transcriptome profiling at crucial stages of the foliage etiolation process. Based on the comprehensive analyses, hormonal balance was disordered, and two rate-limiting genes, NCED3 (9-cis-epoxycarotenoid dioxygenases 3) and NCED5, being responsible for ABA (abscisic acid) accumulation, were highlighted. Further correlation analysis indicated that IAA (indole-3-acetic acid) and ABA were the most likely inducers of the expression of stresses-related genes. In addition, excessive starch accumulation was observed in lamina and midribs of incompatible grafts leaves. These results provided a new insight into the role of the hormonal balance and abscisic acid biosynthesis genes in regulation and contribution to the graft incompatibility, and will further define and deploy candidate genes to explore the mechanisms underlying citrus rootstock- scion interactions.

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Figures

Figure 1
Figure 1
Transcriptome analysis of leaves. a Leaf samples at three phases (P1, P2, P3). b Hierarchical clustering of unigene expression. c Correlation of expression changes observed by RNA-seq (Y-axis) and qPCR (X-axis). d PCA of the samples sequenced by RNA-seq. The X-axis and Y-axes represent the first and second components. Dots with the same color indicate the same graft combination.
Figure 2
Figure 2
DEG analyses. a Venn diagrams of genes differentially expressed between Hm/Pt and controls at the same developmental stage. b KEGG enrichment analysis of the DEGs. c Expression of DEGs in carotenoid biosynthesis and plant hormone signal transduction pathways. Heatmap color indicates FPKM value.
Figure 3
Figure 3
WGCNA of differentially expressed genes. a Hierarchical cluster tree showing co-expression modules identified by WGCNA. Each leaf in the tree is one gene. The major tree branches constitute seven modules labeled with different colors. b KEGG enrichment analysis of the genes in the blue module. c Genes whose expression was highly correlated in the blue module.
Figure 4
Figure 4
Transverse sections showing changes in leaves of compatible and incompatible grafts. a Leaf samples at P3. b Cross-section of leaf. c A midrib section was observed and photographs were taken under a light microscope. d Epifluorescence photomicrographs of phloem. e Starch grains were dyed blue. f, g Contents of starch (f) and soluble sugars (g). h Transcript abundance changes of starch and sucrose metabolism pathways. Asterisks represent significant differences compared with the control (**P < .01), analyzed using Student’s t-test. Heatmap shows the log10 (FPKM + .01) of selected differentially expressed transcripts. Black arrows indicate parenchyma cells and red arrows indicate starch accumulation in c and e.
Figure 5
Figure 5
Working model for graft incompatibility in Hm/Pt. The rootstock–scion interaction induced signals to cause TF gene activity. Differential expression of TFs could directly affect auxin and ABA signal transduction. Genes related to auxin and ABA were differentially expressed, causing a decrease in IAA and an increase in ABA level, and further acclimation to grafting-induced stress, such as water deficiency, starch accumulation and chlorophyll decreased. Metabolic disorder caused structural variation, and led to foliage etiolation. The red and green arrows indicate up- and down-regulation respectively. Square brackets contain reference numbers.
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