IbMPK3/IbMPK6-mediated IbSPF1 phosphorylation promotes tolerance to bacterial pathogen in sweetpotato.

KEY MESSAGE: IbSPF1, a novel target of IbMPK3/IbMPK6, regulates biotic stress response in sweetpotato. Environmental stresses due to biotic and abiotic factors negatively affect crop quality and productivity. To minimize the damage caused by these factors, numerous stress signaling pathways are activated in plants. Among these, the mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in diverse plant stress responses. MPK3 and MPK6 function in several cellular signaling pathways by phosphorylating downstream partner proteins in response to environmental stresses. However, little is known about the MPK3/MPK6 signaling pathway in sweetpotato [Ipomoea batatas (L.) Lam]. We recently confirmed that IbMPK3 and IbMPK6, two pathogen-responsive MAPKs, play essential roles in defense gene activation in sweetpotato. In this study, we show that sweetpotato SP8-binding factor (IbSPF1), a substrate of IbMPK3/IbMPK6, functions as a transcriptional regulator of biotic stress signaling in sweetpotato. IbSPF1 specifically interacts with IbMPK3 and IbMPK6, which phosphorylate Ser75 and Ser110 residues of IbSPF1. This increases the affinity of IbSPF1 for the W-box element in target gene promoters. Additionally, the expression of IbSPF1 was up-regulated under various stress conditions and different hormone treatments involved in plant defense responses. Interestingly, the phospho-mimicking mutant of IbSPF1 showed enhanced resistance to Pseudomonas syringae pv. tabaci, and transient expression of mutant IbSPF1 induced the expression of pathogenesis-related genes. These results indicate that the phosphorylation of IbSPF1 by IbMPK3/IbMPK6 plays a critical role in plant immunity by up-regulating the expression of downstream genes.

Different Functions of IbRAP2.4, a Drought-Responsive AP2/ERF Transcription Factor, in Regulating Root Development Between Arabidopsis and Sweetpotato.

Plant root systems are essential for the uptake of water and nutrients from soil and are positively correlated to yield in many crops including the sweetpotato, Ipomoea batatas (L.) Lam. Here, we isolated and functionally characterized IbRAP2.4, a novel nuclear-localized gene encoding the AP2/ERF transcription factor, from sweetpotato. IbRAP2.4 was responsive to NaCl, PEG8000, ethylene, and Indole 3-acetic acid treatments. As revealed by electrophoretic mobility shift assay and dual luciferase assay, IbRAP2.4 could bind to both DRE and GCC-box elements and acted as a transcription activator. IbRAP2.4 overexpression significantly promoted lateral root formation and enhanced the drought tolerance in Arabidopsis thaliana, while it inhibited storage root formation in transgenic sweetpotato by comprehensively upregulating lignin biosynthesis pathway genes. Results suggested that IbRAP2.4 may be a useful potential target for further molecular breeding of high yielding sweetpotato.

Effects of exogenous phytohormones on chlorogenic acid accumulation and pathway-associated gene expressions in sweetpotato stem tips.

Sweetpotato (Ipomoea batatas [L.] Lam.) stem tips, which contain high concentrations of chlorogenic acid (CGA), are useful as a physiologically functional food to protect against some serious diseases. According to previous studies, exogenous application of phytohormones may be an effective agrotechnical measure to control CGA biosynthesis through the transcriptional regulation of pathway gene expressions. To understand the mechanism of CGA biosynthesis in sweetpotato, we investigated the effects of exogenous phytohormones on CGA metabolism in stem tips of sweetpotato. A significantly elevated CGA content was observed in salicylic acid (SA)-treated sweetpotato stem tips at 72 h, as well as in those subjected to abscisic acid (ABA) or gibberellic acid (GA) treatments. Dynamic expression change of seven enzyme genes involved in sweetpotato CGA biosynthesis were analyzed to determine correlations between transcript levels and CGA accumulation. As revealed by the differential expression of these genes under distinct phytohormone treatments, the regulation of specific pathway genes is a critical determinant of the accumulation of CGA in sweetpotato stem tips. We also found that several hormone-responsive sites, such as those for ABA, GA, SA, and jasmonic acid (JA), were present in the promoter regions of sweetpotato CGA biosynthestic pathway genes. Collectively, phytohormones can regulate the transcription of CGA synthesis-related genes and ultimately affect CGA accumulation in sweetpotato stem tips, whereas the regulatory differences are mirrored by cis-acting elements in the corresponding pathway gene promoters.

Overexpression of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) increases abiotic stress tolerance in transgenic sweetpotato plants.

Tocopherols are lipid-soluble compounds regarded as vitamin E compounds and they function as antioxidants in scavenging lipid peroxyl radicals and quenching reactive oxygen species (ROS). In our previous studies, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants including 4-hydroxyphenylpyruvate dioxygenase (IbHPPD). HPPD is the first regulatory enzyme in vitamin E biosynthesis and serves to catalyze in the first steps α-tocopherol and plastoquinone biosynthesis by converting 4-hydroxyphenylpyruvate (HPP) to homogentisic acid (HGA). In this study, we generated transgenic sweetpotato plants overexpressing IbHPPD under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as HP plants) via Agrobacterium-mediated transformation to understand the function of IbHPPD in sweetpotato. Three transgenic lines (HP3, HP14 and HP15) with high transcript levels of IbHPPD were selected for further characterization. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, after stress treatments, HP plants also showed higher abscisic acid (ABA) contents than NT plants. Under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19-27% in leaves compared with NT plants. These results indicate that increased abiotic stress tolerance in HP plants is related to inducing enhancement of α-tocopherol and ABA contents.

De novo transcriptome sequencing and gene expression profiling of sweet potato leaves during low temperature stress and recovery.

Sweetpotato [Ipomoea batatas (L.) Lam] is an important crop used for food, animal feed, and production of industrial materials. Although it is adapted to a wide range of unfavorable conditions, including drought and high salt, sweetpotato is vulnerable to low temperature, making it difficult to cultivate in low temperature regions. To understand the molecular responses occurring in sweetpotato leaves under low temperature stress, de novo transcriptome assembly was performed in leaves under low temperature stress (LT) and during recovery (RC). In comparison with non-treated controls (NT), 2461 and 1017 differentially expressed genes (DEGs) were identified in LT and RC leaves, respectively. When expression in RC and LT samples was directly compared, 2053 DEGs were detected. To increase understanding of the DEGs, the three datasets were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genome (KEGG) database. The CBF transcriptional cascade, a well-known cold response pathway, was investigated using transcriptomic analysis. In contrast with reports from the cold-tolerant Arabidopsis thaliana, none of the COR genes identified in sweetpotato showed increased expression in response to low temperature. Genes involved in antioxidant enzyme pathways mediating responses to reactive oxygen species (ROS) were investigated during low temperature response. This work provides insight into the molecular basis of the responses of sweetpotato to cold stress. This increased understanding of gene regulation in response to cold stress in sweetpotato will be beneficial for future research into molecular-assisted breeding.

Transgenic sweetpotato plants overexpressing tocopherol cyclase display enhanced α-tocopherol content and abiotic stress tolerance.

Oxidative stress caused by reactive oxygen species (ROS) under various environmental stresses significantly reduces plant productivity. Tocopherols (collectively known as vitamin E) are a group of lipophilic antioxidants that protect cellular components against oxidative stress. Previously, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including tocopherol cyclase (IbTC). In this study, we generated transgenic sweetpotato plants overexpressing IbTC under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as TC plants) via Agrobacterium-mediated transformation to understand the function of IbTC in sweetpotato. Three transgenic lines (TC2, TC9, and TC11) with high transcript levels of IbTC were selected for further characterization. High performance liquid chromatography (HPLC) analysis revealed that α-tocopherol was the most predominant form of tocopherol in sweetpotato tissues. The content of α-tocopherol was 1.6-3.3-fold higher in TC leaves than in non-transgenic (NT) leaves. No significant difference was observed in the tocopherol content of storage roots between TC and NT plants. Additionally, compared with NT plants, TC plants showed enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses, and showed consistently higher levels of photosystem II activity and chlorophyll content, indicating abiotic stress tolerance. These results suggest IbTC as a strong candidate gene for the development of sweetpotato cultivars with increased α-tocopherol levels and enhanced abiotic stress tolerance.

De novo sequencing and comprehensive analysis of the mutant transcriptome from purple sweet potato (Ipomoea batatas L.).

Purple sweet potatoes, rich in anthocyanin, have been widely favored in light of increasing awareness of health and food safety. In this study, a mutant of purple sweet potato (white peel and flesh) was used to study anthocyanin metabolism by high-throughput RNA sequencing and comparative analysis of the mutant and wild type transcriptomes. A total of 88,509 unigenes ranging from 200nt to 14,986nt with an average length of 849nt were obtained. Unigenes were assigned to Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Functional enrichment using GO and KEGG annotations showed that 3828 of the differently expressed genes probably influenced many important biological and metabolic pathways, including anthocyanin biosynthesis. Most importantly, the structural and transcription factor genes that contribute to anthocyanin biosynthesis were downregulated in the mutant. The unigene dataset that was used to discover the anthocyanin candidate genes can serve as a comprehensive resource for molecular research in sweet potato.