Plant leaf patterns and shapes are spectacularly diverse. Changing the complexity of leaflet numbers is a valuable approach to increase its nutrition and photosynthesis. Alfalfa (Medicago sativa) is the most important forage legume species and has diversified compound leaf patterns, which makes it a model species for studying compound leaf development. However, transcriptomic information from alfalfa remains limited. In this study, RNA-Seq technology was used to identify 3746 differentially expressed genes (DEGs) between multifoliate and trifoliate alfalfa. Through an analysis of annotation information and expression data, SPL, one of the key regulators in modifiable plant development and abiotic stress response, was further analyzed. Here, thirty MsSPL genes were obtained from the alfalfa genome, of which 16 had the putative miR156 binding site. A tissue expression pattern analysis showed that the miR156-targeted MsSPLs were divided into two classes, namely, either tissue-specific or widely expressed in all tissues. All miR156-targeted SPLs strongly showed diversification and positive roles under drought and salt conditions. Importantly, miR156/MsSPL08 was significantly suppressed in multifoliate alfalfa. Furthermore, in the paralogous mutant of MsSPL08 isolated from Medicago truncatula, the phenotypes of mutant plants reveal that miR156/MsSPL08 is involved not only involved the branches but also especially regulates the number of leaflets. The legume is a typical compound leaf plant; the ratio of the leaflet often affects the quality of the forage. This study sheds light on new functions of SPL genes that regulate leaflet number development.

Owing to climate change impacts, waterlogging is a serious abiotic stress that affects crops, resulting in stunted growth and loss of productivity. Cassava (Manihot esculentaGrantz) is usually grown in areas that experience high amounts of rainfall; however, little research has been done on the waterlogging tolerance mechanism of this species. Therefore, we investigated the physiological responses of cassava plants to waterlogging stress and analyzed global gene transcription responses in the leaves and roots of waterlogged cassava plants. The results showed that waterlogging stress significantly decreased the leaf chlorophyll content, caused premature senescence, and increased the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the leaves and roots. In total, 2538 differentially expressed genes (DEGs) were detected in the leaves and 13364 in the roots, with 1523 genes shared between the two tissues. Comparative analysis revealed that the DEGs were related mainly to photosynthesis, amino metabolism, RNA transport and degradation. We also summarized the functions of the pathways that respond to waterlogging and are involved in photosynthesis, glycolysis and galactose metabolism. Additionally, many transcription factors (TFs), such as MYBs, AP2/ERFs, WRKYs and NACs, were identified, suggesting that they potentially function in the waterlogging response in cassava. The expression of 12 randomly selected genes evaluated via both quantitative real-time PCR (qRT-PCR) and RNA sequencing (RNA-seq) was highly correlated (R²= 0.9077), validating the reliability of the RNA-seq results. The potential waterlogging stress-related transcripts identified in this study are representatives of candidate genes and molecular resources for further understanding the molecular mechanisms underlying the waterlogging response in cassava.

Phenylalanine ammonia lyase (PAL) is the first enzyme in the phenylpropanoid pathway and plays a critical role in plant growth, development and stress defence. However, there have been few reports of the PAL gene family in lucerne (also known as alfalfa, Medicago sativa L.), one of the most important forage legume species worldwide. In this study, we report that PAL in lucerne is encoded by a family of seven genes: MsPAL1–MsPAL7. Furthermore, a comprehensive genome-wide bioinformatics analysis of the MsPAL gene family is presented, including chromosomal locations, phylogenetic relationships, gene structures and conserved motifs. The cis-elements and potential biological functions of these genes were investigated, revealing the potential roles of MsPAL members in response to various stresses. RT-qPCR results showed that the expression of MsPAL6 was significantly upregulated under both salinity- and waterlogging-stress conditions. Other MsPAL members such as MsPAL1 and MsPAL2 were downregulated under saline conditions and upregulated significantly after waterlogging stress. Our findings provide useful information for further practical analyses and for the genetic improvement of abiotic stress tolerance of lucerne.

Plant-specific TIFY [TIF(F/Y)XG] proteins serve important roles in the regulation of plant stress responses. This family encodes four subfamilies of proteins, JAZ (JASMONATE ZIM-domain), PPD (PEAPOD), ZML (Zinc-finger Inflorescence-like), and TIFY. In this work, a total of 16 JAZ, 3 PPD, 7 ZML, and 2 TIFY genes were found in cassava (Manihot esculenta Crantz) at the genome-wide level. The phylogenetics, exon-intron structure, motif organization, and conserved domains of these genes were analyzed to characterize the members of the JAZ, PPD, and ZML subfamilies. Chromosome location and synteny analyses revealed that 26 JAZ, PPD, and ZML genes were irregularly distributed across 14 of the 18 chromosomes, and 18 gene pairs were implicated in large-scale interchromosomal segmental duplication events. In addition, JAZ, PPD, and ZML gene synteny comparisons between cassava and three other plant species (Arabidopsis, Populus trichocarpa, and rice) uncovered vital information about their likely evolution. The prediction of protein interaction network and cis-acting elements reveal the function of JAZ, PPD, and ZML genes. Subsequently, expression patterns of JAZ, PPD, and ZML genes were validated by qRT-PCR as being expressed in response to osmotic, salt, and cadmium stress. Moreover, almost all JAZ subfamily genes were responsive to jasmonic acid (JA) treatment. In particular, MeJAZ1, MeJAZ13, and MeJAZ14, were highly up-regulated by three treatments, and these genes may deserve further study. This comprehensive study lays the groundwork for future research into TIFY family genes in cassava and may be valuable for genetic improvement of cassava and other related species.

Superoxide dismutase (SOD) is a crucial metal-containing enzyme that plays a vital role in catalyzing the dismutation of superoxide anions, converting them into molecular oxygen and hydrogen peroxide, essential for enhancing plant stress tolerance. We identified 8 SOD genes (4 CSODs, 2 FSODs, and 2 MSODs) in cassava. Bioinformatics analyses provided insights into chromosomal location, phylogenetic relationships, gene structure, conserved motifs, and gene ontology annotations. MeSOD genes were classified into two groups through phylogenetic analysis, revealing evolutionary connections. Promoters of these genes harbored stress-related cis-elements. Duplication analysis indicated the functional significance of MeCSOD2/MeCSOD4 and MeMSOD1/MeMSOD2. Through qRT-PCR, MeCSOD2 responded to salt stress, MeMSOD2 to drought, and cassava bacterial blight. Silencing MeMSOD2 increased XpmCHN11 virulence, indicating MeMSOD2 is essential for cassava’s defense against XpmCHN11 infection. These findings enhance our understanding of the SOD gene family’s role in cassava and contribute to strategies for stress tolerance improvement.