WRKY transcription factors constitute one of the largest plant-specific gene families, regulating various aspects of plant growth, development, physiological processes, and responses to abiotic stresses. This study aimed to comprehensively analyze the WRKY gene family of yam (Dioscorea opposita Thunb.), to understand their expression patterns during the growth and development process and their response to different treatments of yam and analyze the function of DoWRKY71 in detail. A total of 25 DoWRKY genes were identified from the transcriptome of yam, which were divided into six clades (I, IIa, IIc, IId, IIe, III) based on phylogenetic analysis. The analysis of conserved motifs revealed 10 motifs, varying in length from 16 to 50 amino acids. Based on real-time quantitative PCR (qRT-PCR) analysis, DoWRKY genes were expressed at different stages of growth and development and responded differentially to various abiotic stresses. The expression level of DoWRKY71 genes was up-regulated in the early stage and then down-regulated in tuber enlargement. This gene showed responsiveness to cold and abiotic stresses, such as abscisic acid (ABA) and methyl jasmonate (MeJA). Therefore, further study was conducted on this gene. Subcellular localization analysis revealed that the DoWRKY71 protein was localized in the nucleus. Moreover, the overexpression of DoWRKY71 enhanced the cold tolerance of transgenic tobacco and promoted ABA mediated stomatal closure. This study presents the first systematic analysis of the WRKY gene family in yam, offering new insights for studying WRKY transcription factors in yam. The functional study of DoWRKY71 lays theoretical foundation for further exploring the regulatory function of the DoWRKY71 gene in the growth and development related signaling pathway of yam.

Gene expression pattern analysis plays a crucial role in omics research, as it helps us understand the regulatory mechanisms of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method for studying gene expression. Reliable q-PCR results depend on proper data normalization, which requires the use of stable reference genes. Yam, valued as both food and medicine, has significant commercial and economic potential. Its cultivation is being actively promoted in the central and western regions of Inner Mongolia. However, studies have shown that no reference gene is universally stable under all conditions. Therefore, screening and validating suitable reference genes are essential for accurately studying gene expression patterns in yam. This study evaluated nine potential reference genes based on transcriptome data of Chinese yam (Dioscorea opposita Thunb.), under various conditions, including yam tuber development, different tissues, High temperature, Low temperature, salt stress (NaCl), drought stress (PEG), abscisic acid stress (ABA), and methyl jasmonate treatment (MeJA). Using geNorm, NormFinder, BestKeeper, and RefFinder, we ranked these nine reference genes according to their expression stability. The results revealed that EIF was not suitable as a reference gene. Conversely, UBQ and PP2A were identified as the most stable and ideal reference genes for expression analysis across different conditions. Our findings provide a theoretical foundation for selecting reference genes in yam and will support more accurate gene expression studies within the genus Dioscorea.

Temperature affects the growth and yield of yam (Dioscorea opposite Thunb.), and calcium-dependent protein kinases (CDPKs) play an important role in the plant stress response. However, there has been a lack of system analyses of yam’s CDPK gene family. In this study, 29 CDPK transcriptome sequences with complete open reading frames (ORFs) were identified from yam RNA sequencing data. The sequences were classified into four groups (I–VI) using phylogenetic analysis. Two DoCDPK genes were randomly selected from each group and the gene patterns of yam leaves were determined using quantitative real-time PCR (qRT-PCR) under high and low temperature stress in order to show their unique functions in mediating specific responses. Among them, DoCDPK20 was significantly induced in high temperatures. The pPZP221-DoCDPK20 was transformed into tobacco leaves using an agrobacterium-mediated method. Under high temperature stress, DoCDPK20 overexpression reduced photosynthesis and improved heat tolerance in transgenic tobacco. Our research offers meaningful perspectives into CDPK genes and new avenues for the genetic engineering and molecular breeding of yam.

Yam (Dioscorea opposita) tuber development is a complex process regulated by various phytohormones, with gibberellin (GA) playing a crucial role. However, the underlying mechanisms and interaction of GA with other phytohormone pathways on yam tuber development remain incompletely understood. This study investigated the regulatory role of GA and its crosstalk with other phytohormones during yam tuber growth through phenotypic, cytological, physiological, and transcriptomic as well as targeted phytohormone metabolomics analyses. The results reveal that exogenous GA promoted tuber enlargement increases vascular bundle and the number and diameter of sieve tubes, and alters the expression of GA anabolism genes and GA signal transduction pathways. Integrated transcriptome and targeted metabolomics analyses revealed coordinated changes in GA and ethylene (ETH) biosynthesis and signaling pathways during tuber development, particularly DELLA-GAI2 acting as a negative regulator of GA signaling. Overexpression of DoDELLA-GAI2 in transgenic tobacco significantly reduced GA level, starch, cytokinin (CTK), and ETH content, as well as aerenchyma tissue growth and parenchyma cell size. Exogenous GA and ethephon treatments increased GA, starch, CTK, and ETH content, and downregulated DoDELLA-GAI2 gene expression. The yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays confirmed a direct interaction between DoDELLA-GAI2 and DoMTCPB, an upstream gene-encoding key enzyme in ETH biosynthesis. DoDELLA-GAI2 acts as a negative regulator of ETH synthesis by interacting with DoMTCPB. GA-induced degradation of DoDELLA-GAI2 relieves this inhibition, promoting ETH production and contributing to tuber growth. Taken together, our findings reveal a novel mechanism based on DoDELLA-GAI2 integrating the GA and ETH signaling processes to regulate tuber development in D. opposita, offering a potential target for improving yam crop productivity.

Yam (Dioscorea cayennensis subsp. rotundata,hereafter referred to as Dioscorea rotundata) is a staple tropical tuber crop with notable nutritional and economic value. Its development and yield depend on efficient sucrose allocation from source tissues. Sucrose transporters (SUTs), a conserved family of membrane proteins, mediate sucrose loading, translocation, and unloading. Although well-studied in model plants and cereals, SUTs in yam remain largely uncharacterized. This study aims to identify and characterize the SUT gene family in yam and explore their roles in sucrose transport and tuber development. We conducted a genome-wide analysis of yam SUT genes, including gene structure, subcellular localization, and phylogeny. Molecular docking was used to predict sucrose-binding residues, and qRT-PCR assessed gene expression across tissues and tuber developmental stages. Eight SUT genes were identified and classified based on sequence similarity and domain structure. Docking analysis revealed key residues involved in sucrose binding and possible conformational shifts influencing transport. Expression profiling showed that most SUT genes, especially in the tuber apex, were progressively upregulated during development, suggesting roles in sucrose unloading and cell expansion. Additionally, functional validation of DrSUT1 in Arabidopsis thaliana confirmed its involvement in sucrose transport, supporting its role in yam sucrose partitioning. Yam SUT genes, especially those highly expressed in sink tissues, are involved in sucrose partitioning and tuber development. These findings provide structural and functional insights into SUT-mediated sugar transport and lay a foundation for improving sucrose utilization and yield in yam and other tuber crops.