Xiao-Hua Lu

Background: Soluble starch synthases are essential for starch formation, but their transcriptional regulation remains poorly understood in cassava. Starch synthase IV (SSIV) is an important enzyme in starch biosynthesis, and this study aimed to investigate the transcriptional regulation of cassava MeSSIV. Methods: Yeast one-hybrid, transcriptional activation, subcellular localization, and dual luciferase assays were performed to evaluate MeWRKY12 binding to and activation of the MeSSIV promoter. Quantitative real-time PCR was used to analyze MeWRKY12 expression in different tissues. Histochemical staining and quantitative GUS assays were conducted using proMeWRKY12 reporter plants grown under field conditions for six months. Promoter responses to abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA₃) were examined in storage root slices. Results: MeWRKY12 bound to the tested MeSSIV promoter region in yeast, exhibited transcriptional activation activity, localized to the nucleus, and enhanced MeSSIV promoter activity in plant cells. MeWRKY12 transcript abundance was highest in storage roots. GUS activity was also highest in storage roots, followed by fibrous roots. Histochemical staining revealed broad promoter activity in vascular, cambial, and parenchymatous tissues, including the xylem parenchyma of storage roots. Promoter activity increased following ABA, MeJA, and GA₃ treatments, with distinct temporal response patterns. Conclusions: These findings provide evidence at the promoter level supporting MeWRKY12 as a candidate positive regulator of MeSSIV transcription and reveal variation in proMeWRKY12 activity across tissues and hormone treatments. This study provides a basis for investigating WRKY transcription factors in the regulation of starch biosynthesis in cassava.

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the −264 bp to −1 bp MeSSIII-1 promoter has basal activity. The range from −1228 bp to −987 bp and −488 bp to −264 bp significantly enhance promoter activity. The regions from −987 bp to −747 bp and −747 bp to −488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

As a starchy and edible tropical plant, cassava (Manihot esculenta Crantz) has been widely used as an industrial raw material and a dietary source. However, the metabolomic and genetic differences in specific germplasms of cassava storage root were unclear. In this study, two specific germplasms, M. esculenta Crantz cv. sugar cassava GPMS0991L and M. esculenta Crantz cv. pink cassava BRA117315, were used as research materials. Results showed that sugar cassava GPMS0991L was rich in glucose and fructose, whereas pink cassava BRA117315 was rich in starch and sucrose. Metabolomic and transcriptomic analysis indicated that sucrose and starch metabolism had significantly changing metabolites enrichment and the highest degree of differential expression genes, respectively. Sugar transport in storage roots may contribute to the activities of sugar, which will eventually be exported to transporters (SWEETs), such as (MeSWEET1a, MeSWEET2b, MeSWEET4, MeSWEET5, MeSWEET10b, and MeSWEET17c), which transport hexose to plant cells. The expression level of genes involved in starch biosynthesis and metabolism were altered, which may result in starch accumulation. These results provide a theoretical basis for sugar transport and starch accumulation and may be useful in improving the quality of tuberous crops and increasing yield.

Cassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (Manihot esculenta Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with Agrobacterium strain LBA4404 was presented for the first time. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. The transformation efficiency was increased in the conditions of Agrobacterium strain cell infection density (OD₆₀₀ = 0.65), 250 µM acetosyringone induction, and agro-cultivation with wet FECs for 3 days in dark. Based on the optimized transformation protocol, approximately 120–140 independent transgenic lines per mL settled cell volume (SCV) of FECs were created by gene transformation in approximately 5 months, and 45.83% homozygous mono-allelic mutations of the MePDS gene with a YAO promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.

Annexins are a superfamily of soluble calcium-dependent phospholipid-binding proteins that have considerable regulatory effects in plants, especially in response to adversity and stress. The Arabidopsis thaliana AtAnn1 gene has been reported to play a significant role in various abiotic stress responses. In our study, the cDNA of an annexin gene highly similar to AtAnn1 was isolated from the cassava genome and named MeAnn2. It contains domains specific to annexins, including four annexin repeat sequences (I–IV), a Ca²⁺-binding sequence, Ca²⁺-independent membrane-binding-related tryptophan residues, and a salt bridge-related domain. MeAnn2 is localized in the cell membrane and cytoplasm, and it was found to be preferentially expressed in the storage roots of cassava. The overexpression of MeAnn2 reduced the sensitivity of transgenic Arabidopsis to various Ca²⁺, NaCl, and indole-3-acetic acid (IAA) concentrations. The expression of the stress resistance-related gene AtRD29B and auxin signaling pathway-related genes AtIAA4 and AtLBD18 in transgenic Arabidopsis was significantly increased under salt stress, while the Malondialdehyde (MDA) content was significantly lower than that of the control. These results indicate that the MeAnn2 gene may increase the salt tolerance of transgenic Arabidopsis via the IAA signaling pathway.