TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns.

BACKGROUND: Tonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy. RESULTS: In this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1-5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and - 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns. CONCLUSIONS: Our findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut.

Molecular characterization of oleosin genes in Cyperus esculentus, a Cyperaceae plant producing oil in underground tubers.

KEY MESSAGE: CeOLE genes exhibit a tuber-predominant expression pattern and their mRNA/protein abundances are positively correlated with oil accumulation during tuber development. Overexpression could significantly increase the oil content of tobacco leaves. Oleosins (OLEs) are abundant structural proteins of lipid droplets (LDs) that function in LD formation and stabilization in seeds of oil crops. However, little information is available on their roles in vegetative tissues. In this study, we present the first genome-wide characterization of the oleosin family in tigernut (Cyperus esculentus L., Cyperaceae), a rare example accumulating high amounts of oil in underground tubers. Six members identified represent three previously defined clades (i.e. U, SL and SH) or six out of seven orthogroups (i.e. U, SL1, SL2, and SH1-3) proposed in this study. Comparative genomics analysis reveals that lineage-specific expansion of Clades SL and SH was contributed by whole-genome duplication and dispersed duplication, respectively. Moreover, presence of SL2 and SH3 in Juncus effuses implies their appearance sometime before Cyperaceae-Juncaceae divergence, whereas SH2 appears to be Cyperaceae specific. Expression analysis showed that CeOLE genes exhibit a tuber-predominant expression pattern and transcript levels are considerably more abundant than homologs in the close relative Cyperus rotundus. Moreover, CeOLE mRNA and protein abundances were shown to positively correlate with oil accumulation during tuber development. Additionally, two dominant isoforms (i.e. CeOLE2 and -5) were shown to locate in LDs as well as the endoplasmic reticulum of tobacco (Nicotiana benthamiana) leaves, and are more likely to function in homo and heteromultimers. Furthermore, overexpression of CeOLE2 and -5 in tobacco leaves could significantly increase the oil content, supporting their roles in oil accumulation. These findings provide insights into lineage-specific family evolution and putative roles of CeOLE genes in oil accumulation of vegetative tissues, which facilitate further genetic improvement for tigernut.

Analysis of oil synthesis pathway in Cyperus esculentus tubers and identification of oleosin and caleosin genes.

The tubers of the widely distributed Cyperus esculentus are rich in oil, and therefore, the plant is considered to have a high utilization value in the vegetable oil industry. Oleosins and caleosins are lipid-associated proteins found in oil bodies of seeds; however oleosins and caleosins genes have not been identified in C. esculentus. In this study, we performed transcriptome sequencing and lipid metabolome analysis of C. esculentus tubers at four developmental stages to obtain the information on their genetic profile, expression trends, and metabolites in oil accumulation pathways. Overall, 120,881 non-redundant unigenes and 255 lipids were detected; 18 genes belonged to the acetyl-CoA carboxylase (ACC), malonyl-CoA:ACP transacylase (MCAT), ß-ketoacyl-ACP synthase (KAS), and fatty acyl-ACP thioesterase (FAT) gene families involved in fatty acid biosynthesis, and 16 genes belonged to the glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase 3 (DGAT3), phospholipid:diacylglycerol acyltransferase (PDAT), FAD2, and lysophosphatidic acid acyltransferase (LPAAT) gene families playing important roles in triacylglycerol synthesis. We also identified 9 oleosin- and 21 caleosin-encoding genes in C. esculentus tubers. These results provide detailed information on the C. esculentus transcriptional and metabolic profiles, which can be used as reference for the development of strategies to increase oil content in C. esculentus tubers.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

Characterisation of two novel genes encoding Δ9 fatty acid desaturases (CeSADs) for oleic acid accumulation in the oil-rich tuber of Cyperus esculentus.

Cyperus esculentus is considered one of the most promising oil crops due to its oil-rich tuber, wide adaptability and large biomass production. Preferable triacylglycerol (TAG) composition, especially high oleic acid content, makes tuber oil suitable for human consumption and biodiesel production. However, the mechanism underlying oleic acid enrichment in the tuber remains unknown. Plastidial stearoyl-ACP desaturase (SAD) catalyses the formation of monounsaturated fatty acids (MUFAs), which may function crucially for high accumulation of oleic acid in C. esculentus tubers. In this study, two full-length cDNAs encoding SAD were isolated from the developing tubers of C. esculentus, namely, CeSAD1 and CeSAD2, with ORFs of 1194 bp and 1161 bp, respectively. Quantitative RT-PCR analysis showed that CeSAD genes were highly expressed in tubers. The expression pattern during tuber formation was also significantly correlated with fatty acid and oil accumulation dynamics. Overexpression of each CeSAD gene could restore the normal growth of the defective yeast BY4389, indicating that both CeSADs had fatty acid desaturase activity to catalyse MUFA biosynthesis. A tobacco genetic transformation assay demonstrated that both CeSAD enzymes had high enzyme activity. Exogenous addition of exogenous fatty acids to feed yeast revealed that CeSAD1 has a more substantial substrate preference ratio for C18:0 than CeSAD2 did. Moreover, the overexpression of CeSAD1 significantly increased host tolerance against low-temperature stress. Our data add new insights into the deep elucidation of oleic acid-enriched oils in Cyperus esculentus tubers, showing CeSAD, especially CeSAD1, as the target gene in genetic modification to increase oil and oleic yields in oil crops as well as stress tolerance.

Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data.

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

Functional Characterization of Three Novel Genes Encoding Diacylglycerol Acyltransferase (DGAT) from Oil-Rich Tubers of Cyperus esculentus.

Cyperus esculentus is probably the only plant that is known to accumulate large amounts of oil in its tubers. However, the underlying metabolic mechanism and regulatory factors involved in oil synthesis of tubers are still largely unclear. In this study, one gene encoding type I diacylglycerol acyltransferase (DGAT) (CeDGAT1) and two genes encoding type II DGAT (CeDGAT2a and CeDGAT2b) from C. esculentus were identified and functionally analyzed. All three DGAT genes were found to be expressed in tuber, root and leaf tissues. CeDGAT1 is highly expressed in roots and leaves, whereas CeDGAT2b is dominantly expressed in tubers. Furthermore, the temporal expression pattern of CeDGAT2b is well coordinated with the oil accumulation in developing tubers. When each CeDGAT was heterologously expressed in triacylglycerol (TAG)-deficient mutant of Saccharomyces cerevisiae, Arabidopsis thaliana wild type or its TAG1 mutant with AtDGAT1 disruption, only CeDGAT2b showed the ability to restore TAG biosynthesis with lipid body formation in yeast mutant, enhance seed oil production of Arabidopsis wild type and rescue multiple seed phenotypes of TAG1 mutant. In addition, CeDGAT2b was shown to have a substrate preference for unsaturated fatty acids toward TAG synthesis. Taken together, our results indicated that CeDGAT2b from C. esculentus is an actively functional protein and is most likely the major contributor to tuber oil biosynthesis containing common fatty acids, in contrast to oil-rich seeds and fruits where DGAT1 plays a more central role than DGAT2 in oil production accumulating normal fatty acids, whereas DGAT2 is a primary regulator for oil synthesis rich in unusual fatty acids.

Oil Biosynthesis in Underground Oil-Rich Storage Vegetative Tissue: Comparison of Cyperus esculentus Tuber with Oil Seeds and Fruits.

Cyperus esculentus is unique in that it can accumulate rich oil in its tubers. However, the underlying mechanism of tuber oil biosynthesis is still unclear. Our transcriptional analyses of the pathways from pyruvate production up to triacylglycerol (TAG) accumulation in tubers revealed many distinct species-specific lipid expression patterns from oil seeds and fruits, indicating that in C. esculentus tuber: (i) carbon flux from sucrose toward plastid pyruvate could be produced mostly through the cytosolic glycolytic pathway; (ii) acetyl-CoA synthetase might be an important contributor to acetyl-CoA formation for plastid fatty acid biosynthesis; (iii) the expression pattern for stearoyl-ACP desaturase was associated with high oleic acid composition; (iv) it was most likely that endoplasmic reticulum (ER)-associated acyl-CoA synthetase played a significant role in the export of fatty acids between the plastid and ER; (v) lipid phosphate phosphatase (LPP)-δ was most probably related to the formation of the diacylglycerol (DAG) pool in the Kennedy pathway; and (vi) diacylglyceroltransacylase 2 (DGAT2) and phospholipid:diacylglycerolacyltransferase 1 (PDAT1) might play crucial roles in tuber oil biosynthesis. In contrast to oil-rich fruits, there existed many oleosins, caleosins and steroleosins with very high transcripts in tubers. Surprisingly, only a single ortholog of WRINKLED1 (WRI1)-like transcription factor was identified and it was poorly expressed during tuber development. Our study not only provides insights into lipid metabolism in tuber tissues, but also broadens our understanding of TAG synthesis in oil plants. Such knowledge is of significance in exploiting this oil-rich species and manipulating other non-seed tissues to enhance storage oil production.

Transcriptional transitions in Nicotiana benthamiana leaves upon induction of oil synthesis by WRINKLED1 homologs from diverse species and tissues.

BACKGROUND: Carbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. Knowledge about the regulation of carbon allocation into oil (triacylglycerol) in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. Here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor WRINKLED1 (WRI1) originating from diverse species and tissues; Arabidopsis thaliana and potato (Solanum tuberosum) seed embryo, poplar (Populus trichocarpa) stem cambium, oat (Avena sativa) grain endosperm, and nutsedge (Cyperus esculentus) tuber parenchyma, were studied by agroinfiltration in Nicotiana benthamiana. RESULTS: All WRI1 homologs induced oil accumulation when expressed in leaf tissue. Transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: Transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. Transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. Moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle. RT-qPCR analysis of leaves expressing Arabidopsis WRI1 showed the temporal trends of transcripts selected as 'markers' for key metabolic pathways one to five days after agroinfiltration. Chlorophyll fluorescence measurements of leaves expressing Arabidopsis WRI1 showed a significant decrease in photosynthesis, even though effect on starch content could not be observed. CONCLUSIONS: This data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by WRI1. This yields important information about what effects WRI1 may exert on global gene expression during seed and embryo development. The results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by WRI1, which can be essential knowledge in the development of new high-yielding oil crops.

Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge).

ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the first committed step of starch synthesis by catalyzing the biosynthesis of ADP-glucose from glucose-phosphate and ATP. It is a tetrameric protein consisting of two small and two large subunits. The small subunits have a catalytic function, while the large subunits regulate the enzyme activity. Cyperus esculentus (yellow nutsedge) is a perennial C4 plant grown from rhizomes and tubers. Previous studies on yellow nutsedge have mostly focused on the morphology and cultivation of tubers, their application in food, and biochemical analyses of the tubers. In this study, the gene encoding the ADPGlcPPase small subunit (CeAGPS) in yellow nutsedge was cloned and characterized. The full-length CeAGPS cDNA sequence contained an 81-bp 5'-untranslated region (UTR), a 188-bp 3'-UTR, and a 1539-bp open reading frame encoding 512-amino acid residues. The genomic sequence of CeAGPS comprises a nine exon-eight intron structure similar to the previously reported cotton and Arabidopsis thaliana AGPS genes. The deduced translation product of the CeAGPS gene contained a well-conserved catalytic domain and regulatory elements typical of plant AGPS. Reverse transcriptase polymerase chain reaction amplification of the target gene in various plant parts using gene-specific primers indicated that the expression of CeAGPS was most abundant in the tuber, and relatively lower in nutsedge roots.