Integrated Characterization of Cassava (Manihot esculenta) Pectin Methylesterase (MePME) Genes to Filter Candidate Gene Responses to Multiple Abiotic Stresses.

Plant pectin methylesterases (PMEs) play crucial roles in regulating cell wall modification and response to various stresses. Members of the PME family have been found in several crops, but there is a lack of research into their presence in cassava (Manihot esculent), which is an important crop for world food security. In this research, 89 MePME genes were identified in cassava that were separated into two types (type-Ⅰ and type-Ⅱ) according to the existence or absence of a pro-region (PMEI domain). The MePME gene members were unevenly located on 17 chromosomes, with 19 gene pairs being identified that most likely arose via duplication events. The MePMEs could be divided into ten sub-groups in type-Ⅰ and five sub-groups in type-Ⅱ. The motif analysis revealed 11 conserved motifs in type-Ⅰ and 8 in type-Ⅱ MePMEs. The number of introns in the CDS region of type-Ⅰ MePMEs ranged between one and two, and the number of introns in type-Ⅱ MePMEs ranged between one and nine. There were 21 type-Ⅰ and 31 type-Ⅱ MePMEs that contained signal peptides. Most of the type-Ⅰ MePMEs had two conserved "RK/RLL" and one "FPSWVS" domain between the pro-region and the PME domain. Multiple stress-, hormone- and tissue-specific-related cis-acting regulatory elements were identified in the promoter regions of MePME genes. A total of five co-expressed genes (MePME1, MePME2, MePME27, MePME65 and MePME82) were filtered from different abiotic stresses via the use of UpSet Venn diagrams. The gene expression pattern analysis revealed that the expression of MePME1 was positively correlated with the degree of cassava postharvest physiological deterioration (PPD). The expression of this gene was also significantly upregulated by 7% PEG and 14 °C low-temperature stress, but slightly downregulated by ABA treatment. The tissue-specific expression analysis revealed that MePME1 and MePME65 generally displayed higher expression levels in most tissues than the other co-expressed genes. In this study, we obtain an in-depth understanding of the cassava PME gene family, suggesting that MePME1 could be a candidate gene associated with multiple abiotic tolerance.

Changes in sucrose metabolism patterns affect the early maturation of Cassava sexual tetraploid roots.

BACKGROUND: Cassava (Manihot esculenta Crantz) is an important multiuse crop grown for economic and energy purposes. Its vegetative organs are storage roots, in which the main storage material is starch. The accumulation characteristics of starch in cassava roots can directly affect the yield, starch content and maturation of cassava storage roots. In this study, we used a cassava sexual tetraploid (ST), which showed early maturation heterosis in previous work, as the main test material. We analyzed the sucrose metabolism and starch accumulation characteristics of the ST and its parents from the leaf "source" to the storage root "sink" during different developmental stages and explored the regulatory mechanisms of ST storage root early maturation by combining the transcriptome data of the storage roots during the expansion period. RESULTS: The results showed that the trends in sucrose, glucose and fructose contents in the ST leaves were similar to those of the two parents during different stages of development, but the trends in the ST storage roots were significantly different from those of their parents, which showed high sucrose utilization rates during the early stage of development and decreased utilization capacity in the late developmental stage. Transcriptome data showed that the genes that were expressed differentially between ST and its parents were mainly involved in the degradation and utilization of sucrose in the storage roots, and four key enzyme genes were significantly upregulated (Invertase MeNINV8/MeVINV3, Sucrose synthase MeSuSy2, Hexokinase MeHXK2), while the expressions of key enzyme genes involved in starch synthesis were not significantly different. CONCLUSIONS: The results revealed that the pattern of sucrose degradation and utilization in the cassava ST was different from that of its parents and promoted early maturation in its tuberous roots. Starch accumulation in the ST from sucrose mainly occurred during the early expansion stage of the storage roots, and the starch content during this period was higher than that of both parents, mainly due to the regulation of invertase and hexokinase activities during sucrose metabolism. This study provides a basis for further genetic improvements to cassava traits and for breeding varieties that mature early and are adapted well to provide starch supply requirements.

Overexpression of MePMEI1 in Arabidopsis enhances Pb tolerance.

Pb is one of the most ubiquitously distributed heavy metal pollutants in soils and has serious negative effects on plant growth, food safety, and public health. Pectin methylesterase inhibitors (PMEIs) play a pivotal role in regulating the integrity of plant cell walls; however, the molecular basis by which PMEIs promote plant resistance to abiotic stress remains poorly understood. In this study, we identified a novel PMEI gene, MePMEI1, from Manihot esculenta, and determined its role in plant resistance to Pb stress. The expression of MePMEI1 was remarkably upregulated in the roots, stems, and leaves of cassava plants following exposure to Pb stress. An analysis of subcellular localization revealed that the MePMEI1 protein was localized in the cell wall. MePMEI1 inhibited commercial orange peel pectin methyltransferase (PME), and the expression of MePMEI1 in Arabidopsis decreased the PME activity, indicating that MePMEI1 can inhibit PME activity in the cell wall. Additionally, the overexpression of MePMEI1 in Arabidopsis reduced oxidative damage and induced the thickening of cell walls, thus contributing to Pb tolerance. Altogether, the study reports a novel mechanism by which the MePMEI1 gene, which encodes the PMEI protein in cassava, plays an essential role in promoting tolerance to Pb toxicity by regulating the thickness of cell walls. These results provide a theoretical basis for the MePMEI1-mediated plant breeding for increasing heavy metal tolerance and provide insights into controlling Pb pollution in soils through phytoremediation in future studies.

Genome-Wide Identification of Cassava Glyoxalase I Genes and the Potential Function of MeGLYâ -13 in Iron Toxicity Tolerance.

Glyoxalase I (GLYI) is a key enzyme in the pathway of the glyoxalase system that degrades the toxic substance methylglyoxal, which plays a crucial part in plant growth, development, and stress response. A total of 19 GLYI genes were identified from the cassava genome, which distributed randomly on 11 chromosomes. These genes were named MeGLYI-1-19 and were systematically characterized. Transcriptome data analysis showed that MeGLYIs gene expression is tissue-specific, and MeGLYI-13 is the dominant gene expressed in young tissues, while MeGLYI-19 is the dominant gene expressed in mature tissues and organs. qRT-PCR analysis showed that MeGLYI-13 is upregulated under 2 h excess iron stress, but downregulated under 6, 12, and 20 h iron stress. Overexpression of MeGLYI-13 enhanced the growth ability of transgenic yeast under iron stress. The root growth of transgenic Arabidopsis seedlings was less inhibited by iron toxicity than that of the wild type (WT). Potted transgenic Arabidopsis blossomed and podded under iron stress, but flowering of the WT was significantly delayed. The GLYI activity in transgenic Arabidopsis was improved under both non-iron stress and iron stress conditions compared to the WT. The SOD activity in transgenic plants was increased under iron stress, while the POD and CAT activity and MDA content were decreased compared to that in the WT. These results provide a basis for the selection of candidate genes for iron toxicity tolerance in cassava, and lay a theoretical foundation for further studies on the functions of these MeGLYI genes.

MeNINV1: An Alkaline/Neutral Invertase Gene of Manihot esculenta, Enhanced Sucrose Catabolism and Promoted Plant Vegetative Growth in Transgenic Arabidopsis.

Alkaline/neutral invertase (A/N-INV) is an invertase that irreversibly decomposes sucrose into fructose as well as glucose and plays a role in plant growth and development, starch synthesis, abiotic stress, and other plant-life activities. Cassava is an economically important starch crop in tropical regions. During the development of cassava tuber roots, A/N-INV activity is relatively high, which indicates that it may participate in sucrose metabolism and starch synthesis. In this study, MeNINV1 was confirmed to function as invertase to catalyze sucrose decomposition in yeast. The optimal enzymatic properties of MeNINV1 were a pH of 6.5, a reaction temperature of 40 °C, and sucrose as its specific catalytic substrate. VB6, Zn2+, and Pb2+ at low concentrations as well as EDTA, DTT, Tris, Mg2+, and fructose inhibited A/N-INV enzymic activity. In cassava, the MeNINV1 gene was mainly expressed in the fibrous roots and the tuber root phloem, and its expression decreased as the tuber root grew. MeNINV1 was confirmed to localize in chloroplasts. In Arabidopsis, MeNINV1-overexpressing Arabidopsis had higher A/N-INV activity, and the increased glucose, fructose, and starch content in the leaves promoted plant growth and delayed flowering time but did not change its resistance to abiotic stress. Our results provide new insights into the biological function of MeNINV1.

Comparative Transcriptome Profiling of Cassava Tuberous Roots in Response to Postharvest Physiological Deterioration.

Cassava is one of the most versatile tuberous-root crops on Earth. However, the postharvest storage properties of cassava tuberous root mean that it is perishable through a process known as postharvest physiological deterioration (PPD), which seriously affects its starch quality. Therefore, a comprehensive understanding of the transcriptional regulatory activity of cassava against the PPD response is necessary in order to extract key molecular mechanisms related to PPD tolerance. In this study, we found that RYG1 tuberous roots showed delayed PPD compared to those of SC8. In addition, RYG1 roots maintained a more stable cell wall structure after storage than those of SC8. The transcriptome changes in tuberous roots were analyzed for both RYG1 and SC8 after 21 days of storage (SR and SS) compared to fresh (FR and FS) by the RNA-Seq method. The total number of differentially expressed genes (DEGs) in the various comparisons of these four samples ranged from 68 to 3847. Of these, a total of 2008 co-DEGs in SR vs. SS were shared by either SR vs. FR or SS vs. FS. GO and KEGG enrichment analysis revealed that upregulated co-DEGs in SR vs. SS were mainly enriched in photosynthesis, protein processing, hormone and cutin, suberine and wax biosynthesis. By contrast, the downregulated co-DEGs were mainly related to cell wall organization, starch and sucrose metabolism, galactose metabolism, phenylpropanoid biosynthesis, diterpenoid biosynthesis, cysteine and methionine metabolism and flavonoid biosynthesis. The protein-protein interaction (PPI) networks of the co-DEGs showed a complex interaction of genes in different pathways, and 16 hub genes were characterized to have a degree in excess of 15, among which eight genes were associated with photosynthesis. These results provide new information for the study of cassava resistance to PPD and lay a foundation for the further molecular breeding of storage-tolerant cassava varieties.

Overexpression of Cassava MeAnn2 Enhances the Salt and IAA Tolerance of Transgenic Arabidopsis.

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.