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.

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

The characterization of SaPIN2b, a plant trichome-localized proteinase inhibitor from Solanum americanum.

Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.

Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes.

In this paper, we report a useful protocol for cloning toxic protein genes. Use of the SOC medium, which is a glucose-containing rich medium, significantly improved the transformation efficiency of a recombinant plasmid containing a toxic plant subtilase SaSBT1 cDNA. Both glucose and rich nutrients present in the SOC medium prevented the unintended activation of the lac promoter carried on the cloning vector, and led to significantly improved transformation efficiency of recombinant plasmids containing toxic protein genes and an increased rate of transformant growth.

Overexpression of a weed (Solanum americanum) proteinase inhibitor in transgenic tobacco results in increased glandular trichome density and enhanced resistance to Helicoverpa armigera and Spodoptera litura.

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

Genome sequence and characterization of a new virus infecting Mikania micrantha H.B.K.

The complete RNA genomic sequence of a new virus infecting Mikania micrantha, designated as Mikania micrantha wilt virus (MMWV), has been determined. The genomic sequence and the predicted gene products of MMWV were similar to those of the other viruses of the genus Fabavirus. The MMWV nucleotide sequence showed 75.6% identity to that of gentian mosaic virus, 56.6 and 57% identity to those of two Broad bean wilt virus 1 isolates, and between 55.7 and 58% identity to those of seven Broad bean wilt virus 2 isolates. Our results suggested that MMWV represents a distinct isolate of the candidate species Gentian mosaic virus.

[Oil contents and fatty acid composition in Jatropha curcas seeds collected from different regions].

OBJECTIVE: To compare the oil contents and fatty acid composition among the samples of Jatropha curcas L. seeds collected from China (Guangdong, Hainan, and Guizhou Provinces) and India. METHODS: Soxhlet extraction method and gas chromatography-mass spectrometry (GC-MS) were employed to determine the oil contents of Jatropha seeds and the fatty acid composition of Jatropha oil. RESULTS: The seed oil contents (dry basis) were 32.43% (Guangdong), 31.41% (Hainan), 37.56% (Guizhou) and 41.04% (India), respectively. Twelve different fatty acids were detected by GC-MS, and the content of total unsaturated fatty acids accounted for 80.93%, 79.53%, 77.24% and 78.22% of the total fatty acids in the samples collected from Guangdong, Hainan, Guizhou and India, respectively. CONCLUSION: There are differences in the oil contents and fatty acid composition among the J. curcas seeds collected from different regions, and attention should be given to these differences in the introduction and breeding of J. curcas.

Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate.

The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol-silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate.

Plant bioreactors for pharmaceuticals.

Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. Various plant expression systems or platforms have been tested with certain degrees of success over the past years. However, further development and improvement are needed for more effective plant bioreactors. In this review we first summarize recent progress in various plant bioreactor expression systems and then focus on discussing protein compartmentation to unique organelles and various strategies for developing better plant bioreactors.

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor.

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

The nightshade proteinase inhibitor IIb gene is constitutively expressed in glandular trichomes.

The best known property of plant proteinase inhibitor II (PIN2) genes is their wound-inducible expression in leaves and constitutive expression in flowers. Here we show by promoter analysis in transgenic plants and in situ reverse transcription-PCR (RT-PCR) analysis that SaPIN2b, a member of the PIN2 gene family of nightshade (Solanum americanum), is also constitutively expressed in glandular trichomes. SaPIN2b promoter and its deletions were cloned and fused upstream of beta-glucuronidase (GUS) to transform the nightshade and tobacco (Nicotiana tabacum) plants. Histochemical staining assays indicated that SaPIN2b:GUS was expressed constitutively in glandular trichomes, predominantly in the gland cells, of both transgenic nightshade and tobacco plants. Constitutive expression of SaPIN2b in glandular trichomes was further confirmed by liquid phase in situ RT-PCR analysis of nightshade leaves. Deletion analysis from the 5' end of the SaPIN2b promoter revealed that separate regulatory elements control SaPIN2b expression in gland cells and stalk cells of glandular trichomes. Fluorometric GUS assays showed that SaPIN2b:GUS expression was significantly increased in transgenic plant leaves after mechanical wounding or methyl jasmonate treatment. The SaPIN2b promoter sequence contains six MYB-binding motifs and an L1 box that are involved in trichome differentiation and development. Overexpression of SaPIN2b in tobacco resulted in a significant increase in glandular trichome density and promotion of trichome branching. These results suggest that, as well as being an induced defensive protein of the well-known PIN2 family, SaPIN2b could also play roles in trichome-based defense by functioning as a constitutive component of trichome chemical defense and/or by regulating the development of glandular trichomes.

G-box binding coincides with increased Solanum melongena cysteine proteinase expression in senescent fruits and circadian-regulated leaves.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321-327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at -85/-80 by DNase I footprinting analysis of the -221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.