Summary: Aluminium (Al) toxicity decreases crop production in acid soils in general, but many crops have evolved complex mechanisms to resist it. However, our current understanding of how plants cope with Al stress and perform Al resistance is still at the initial stage. In this study, the citrate transporter CcMATE35 was identified to be involved in Al stress response. The release of citrate was increased substantially in CcMATE35 over‐expression (OE) lines under Al stress, indicating enhanced Al resistance. It was demonstrated that transcription factor CcNFYB3 regulated the expression of CcMATE35, promoting the release of citrate from roots to increase Al resistance in pigeon pea. We also found that a Long noncoding RNA Targeting Citrate Synthase (CcLTCS) is involved in Al resistance in pigeon pea. Compared with controls, overexpression of CcLTCS elevated the expression level of the Citrate Synthase gene (CcCS), leading to increases in root citrate level and citrate release, which forms another module to regulate Al resistance in pigeon pea. Simultaneous overexpression of CcNFYB3 and CcLTCS further increased Al resistance. Taken together, these findings suggest that the two modules, CcNFYB3‐CcMATE35 and CcLTCS‐CcCS, jointly regulate the efflux and synthesis of citrate and may play an important role in enhancing the resistance of pigeon pea under Al stress.

Multidrug and Toxic Compound Extrusion (MATEs) is one of the characteristic transporter families, which plays a key role in the detoxication of endogenous secondary metabolites and exogenous agents in both animal and plant cells. In this study, we identified a total of 67 MATE genes (CcMATEs) from the pigeonpea genome, on which we performed bioinformatics analysis and we group them by phylogenetic analysis. Finally, eight represented CcMATE genes were selected for further qRT-PCR analysis of tissue specificity and response to metal stress in pigeonpea. The results showed that both CcMATE34 and 45 genes were significantly up-regulated and the CcMATE4 gene was only up-regulated in the roots under the stress of Al, Mn and Zn. We speculated that the function of CcMATE34 and 45 might be related to the transport of alkaloids and harmful substances and the function of CcMATE4 might be related to the delivery of flavonoids.

Pigeonpea is an important economic crop in the world and is mainly distributed in tropical and subtropical regions. In order to further expand the scope of planting, one of the problems that must be solved is the impact of soil acidity on plants in these areas. Based on our previous work, we constructed a time series RNA sequencing (RNA-seq) analysis under aluminum (Al) stress in pigeonpea. Through a comparison analysis, 11,425 genes were found to be differentially expressed among all the time points. After clustering these genes by their expression patterns, 12 clusters were generated. Many important functional pathways were identified by gene ontology (GO) analysis, such as biological regulation, localization, response to stimulus, metabolic process, detoxification, and so on. Further analysis showed that metabolic pathways played an important role in the response of Al stress. Thirteen out of the 23 selected genes related to flavonoids and phenols were downregulated in response to Al stress. In addition, we verified these key genes of flavonoid- and phenol-related metabolism pathways by qRT-PCR. Collectively, our findings not only revealed the regulation mechanism of pigeonpea under Al stress but also provided methodological support for further exploration of plant stress regulation mechanisms.