Comparative Transcriptome Analysis of Waterlogging-Sensitive and Tolerant Zombi Pea (Vigna Vexillata) Reveals Energy Conservation and Root Plasticity Controlling Waterlogging Tolerance

Abstract

Vigna vexillata (zombi pea) is an underutilized legume crop considered to be a potential gene source in breeding for abiotic stress tolerance. This study focuses on the molecular characterization of mechanisms controlling waterlogging tolerance using two zombi pea varieties with contrasting waterlogging tolerance. Morphological examination revealed that in contrast to the sensitive variety, the tolerant variety was able to grow, maintain chlorophyll, form lateral roots, and develop aerenchyma in hypocotyl and taproots under waterlogging. To find the mechanism controlling waterlogging tolerance in zombi pea, comparative transcriptome analysis was performed using roots subjected to short-term waterlogging. Functional analysis indicated that glycolysis and fermentative genes were strongly upregulated in the sensitive variety, but not in the tolerant one. In contrast, the genes involved in auxin-regulated lateral root initiation and formation were expressed only in the tolerant variety. In addition, cell wall modification, aquaporin, and peroxidase genes were highly induced in the tolerant variety under waterlogging. Our findings suggest that energy management and root plasticity play important roles in mitigating the impact of waterlogging in zombi pea. The basic knowledge obtained from this study can be used in the molecular breeding of waterlogging-tolerant legume crops in the future.

Keywords: De novo transcriptome; Vigna vexillata; lateral root; legume; waterlogging.

Figure 1

Contrasting waterlogging tolerance in “A408” and “Bali” varieties. Representative 15-day-old zombi pea seedlings subjected to 0, 7, and 10 days of waterlogging. (A) “A408”. (B) “Bali”. Leaf chlorophyll measurement (n = 12 plants) under no stress (NS) and waterlogging (WS). (C) “A408”. (D) “Bali”. * p < 0.05, ** p < 0.01 (t-test).

Figure 2

Changes of zombi pea root architecture under WS. (A) Roots of control plants kept for 7 days under NS. (B). Roots of 7-day WS plants.

Figure 3

Waterlogging induces aerenchyma and extra-cellular airspace in hypocotyls and roots of “A408”. Cross-section of “A408” and “Bali” (A) taproot and (B) hypocotyl. co = cortex. st = stele.

Figure 4

Waterlogging altered root transcriptomes of “A408” and “Bali”. (A) The number of upregulated and downregulated differentially-expressed genes (DEGs) from roots of “A408” and “Bali” in response to WS. (B) Enrichment of GO terms from upregulated and downregulated DEGs from roots of “A408” and “Bali” in response to WS.

Figure 5

Comparative transcriptome response for selected functional categories to WS in roots of “A408” and “Bali”. Over-representation analysis of the DEGs (FDR < 0.05). The statistical analysis of overrepresented functional categories was performed using Fisher method. Z-scores indicate over/under representation. (Number indicates z-score; Yellow indicates over-representation). Data used to generate this figure can be found in Table S4.

Figure 6

Waterlogging caused differential expression of major carbohydrate metabolism, glycolysis, and fermentative genes in roots of “A408” and “Bali”. The number indicates log₂ fold changes. Blue indicates down-regulation. Yellow indicates up-regulation. Data can be found in Table S3.

Figure 7

Differential expression pattern of ethylene synthesis, perception and transcriptional regulator genes in roots of “A408” and “Bali” subjected to WS. The number indicates log₂ fold changes. Blue indicates down-regulation. Yellow indicates up-regulation. Data can be found in Table S3.

Figure 8

Differential expression pattern of auxin metabolism and transcriptional regulator genes in roots of “A408” and “Bali” subjected to WS. The number indicates log₂ fold changes. Blue indicates down-regulation. Yellow indicates up-regulation. Data can be found in Table S3.

Figure 9

Differential expression of transport genes in roots of “A408” and “Bali” subjected to WS. Graphical representation of WS-regulated transport genes based on their assigned protein families. “Up” and “Down” represent up-regulation and down-regulation in this analysis. (A) “A408”. (B) “Bali”. (C) Expression patterns of major intrinsic protein (aquaporin) genes in roots of “A408” and “Bali” under WS. The number indicates log₂ fold changes. Blue indicates down-regulation. Yellow indicates up-regulation. Data can be found in Table S3.

Figure 10

Differential expression of cell wall-related and peroxidase genes in roots of “A408” and “Bali” subjected to WS. Graphical representation of WS-regulated cell wall-related genes based on their assigned protein families. “Up” and “Down” represent up-regulation and down-regulation in this analysis. (A) “A408”. (B) “Bali”. (C) Expression patterns of peroxidase genes in roots of “A408” and “Bali” under WS. The number indicates log₂ fold changes. Blue indicates down-regulation. Yellow indicates up-regulation. Data can be found in Table S3.