The plant-specific RWP-RK transcription factor family plays a central role in the regulation of nitrogen response and gametophyte development. However, little information is available regarding the evolutionary relationships and characteristics of the RWP-RK family genes in cassava, an important tropical crop. Herein, 13 RWP-RK proteins identified in cassava were unevenly distributed across 9 of the 18 chromosomes (Chr), and these proteins were divided into two clusters based on their phylogenetic distance. The NLP subfamily contained seven cassava proteins including GAF, RWP-RK, and PB1 domains; the RKD subfamily contained six cassava proteins including the RWP-RK domain. Genes of the NLP subfamily had a longer sequence and more introns than the RKD subfamily. A large number of hormone- and stress-related cis-acting elements were found in the analysis of RWP-RK promoters. Real-time quantitative PCR revealed that all MeNLP1-7 and MeRKD1/3/5 genes responded to different abiotic stressors (water deficit, cold temperature, mannitol, polyethylene glycol, NaCl, and H₂O₂), hormonal treatments (abscisic acid and methyl jasmonate), and nitrogen starvation. MeNLP3/4/5/6/7 and MeRKD3/5, which can quickly and efficiently respond to different stresses, were found to be important candidate genes for further functional assays in cassava. The MeRKD5 and MeNLP6 proteins were localized to the cell nucleus in tobacco leaf. Five and one candidate proteins interacting with MeRKD5 and MeNLP6, respectively, were screened from the cassava nitrogen starvation library, including agamous-like mads-box protein AGL14, metallothionein 2, Zine finger FYVE domain containing protein, glyceraldehyde-3-phosphate dehydrogenase, E3 Ubiquitin-protein ligase HUWE1, and PPR repeat family protein. These results provided a solid basis to understand abiotic stress responses and signal transduction mediated by RWP-RK genes in cassava.

Shaker K⁺ channel proteins are responsible for potassium (K⁺) uptake and transport, playing a critical role in plant growth, development, and adaptation to K⁺ deficiency. Cassava, a key tropical root crop, is known for its characteristic of resilience to nutrient-poor soil and drought stress. However, the Shaker K⁺ channel gene family in cassava has not yet been characterized. In this study, 13 Shaker channel genes were identified from the near telomere-to-telomere (T2T) cassava genome using bioinformatics analysis. Phylogenetic relationships classified these genes into five distinct subfamilies, and all encoded proteins contained the conserved GYGD/GYGE motif typical of Shaker channels. Protein interaction network predictions revealed potential interactions among the Shaker family, as well as with the potassium transporter HAK5. Tissue-specific expression pattern analysis showed that MeGORK and MeAKT1.2 were expressed in all tissues. Furthermore, quantitative real-time PCR (qRT-PCR) analysis was conducted to examine the transcriptional levels of Shaker K⁺ channel gene family members in the roots and leaves of two cassava germplasms with different low-potassium tolerance after one month of low-potassium treatment. The results revealed that MeAKT1.2, MeAKT2.2, and MeKAT1 exhibited distinct expression patterns between the two germplasms, with higher expression levels observed in the potassium-tolerant germplasm. Therefore, these three genes may serve as important candidate genes for potassium stress tolerance in cassava. In summary, this study provides valuable insights into the characteristics and biological functions of the Shaker K⁺ channel gene family in cassava and identifies potential candidate genes for breeding or engineering potassium-efficient cassava cultivars.

Cassava (Manihot esculenta Crantz) is a major dual-purpose crop in tropical and subtropical regions, but its growth and yield are significantly constrained by abiotic stresses. Calmodulin-binding transcription activators (CAMTAs) are key regulators involved in plant development and stress responses. In this research, six CAMTA genes (MeCAMTAs) were identified from the first telomere-to-telomere (T2T) genome assembly of cassava, and these genes are distributed on four chromosomes. These genes are divided into three different subfamilies based on phylogenetic relationships. Homology analysis shows that there is one pair of replication gene pairs. Analysis of cis-acting elements reveals that the promoter regions of the MeCAMTA gene family contain cis-acting elements responsive to hormones, abiotic stresses, growth and development, and light. Through analysis of the expression patterns of MeCAMTAs in different tissues, MeCAMTAs are expressed in all tissues, among which MeCAMTA3 and MeCAMTA4.1 are mainly expressed in stems, while MeCAMTA1, MeCAMTA2, MeCAMTA4.2, and MeCAMTA6 are mainly expressed in roots. qRT-PCR analysis shows that MeCAMTAs exhibit dynamic expression patterns under different abiotic stress treatments. Therefore, this study provides a certain reference basis for the research on the abiotic stress response mechanism of cassava and also provides potential genetic resources for the stress-resistant breeding of cassava.