Maize Golden2-like transcription factors boost rice chloroplast development, photosynthesis, and grain yield.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra.

Cleome spinosa is widely used as a garden ornamental in many countries. Here we determined the optimal conditions for plant regeneration from different tissue explants grown in vitro. Induction medium containing MS salts, MS vitamins, 3% sucrose, 1 mg l⁻¹ BA, 200 mg l⁻¹ timentin, and 0.8% agar was sufficient for shoot regeneration of all the tissue explants examined, including leaf, hypocotyl, and cotyledon. Subsequently, an Agrobacterium tumefaciens-mediated method was developed to transform the vector pCHS, which carries the transgenes Petunia chalcone synthase (chs) and selection marker neomycin phosphotransferase II (nptII), into C. spinosa. From a total of 368 cotyledon explants, 13 putative transgenic lines were regenerated from selection medium supplemented with 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ timentin, and transferred to the greenhouse. Genomic PCR and Southern blot analyses revealed that the nptII transgene was present in all 13 transgenic plants. Similarly, when the Petunia chs transgene was used as a probe in Southern blot analysis, single or multiple hybridization bands were detected in 12 out of the 13 transgenic plants. In addition, T1 progeny assay from selected transformants showed that the nptII transgene can be transmitted in a Mendelian manner from transgenic parents into their progeny. This is the first report of stable transformation of the C3 dicotyledon C. spinosa, which will facilitate functional comparison of cell-type specific genes with counterpart C4 dicotyledon C. gynandra using transgenic approaches.

Induction of Polyploidy and Metabolic Profiling in the Medicinal Herb Wedelia chinensis.

Wedelia chinensis, which belongs to the Asteraceae family, is a procumbent, perennial herb. It has medicinal anti-inflammatory properties and has been traditionally used as folk medicine in East and South Asia for treating fever, cough and phlegm. In Taiwan, W. chinensis is a common ingredient of herbal tea. Previous studies showed that the plant leaves contain four major bioactive compounds, wedelolactone, demethylwedelolactone, luteolin and apigenin, that have potent antihepatoxic activity, and are thus used as major ingredients in phytopharmaceutical formulations. In this study, we set up optimal conditions for induction of ploidy in W. chinensis. Ploidy can be an effective method of increasing plant biomass and improving medicinal and ornamental characteristics. By using flow cytometry and chicken erythrocyte nuclei as a reference, the DNA content (2C) or genome size of W. chinensis was determined to be 4.80 picograms (pg) in this study for the first time. Subsequently, we developed the successful induction of five triploid and three tetraploid plants by using shoot explants treated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 g/L) of colchicine. No apparent morphological changes were observed between these polyploid plants and the diploid wild-type (WT) plant, except that larger stomata in leaves were found in all polyploid plants as compared to diploid WT. Ultra-performance liquid chromatography coupled with tandem mass spectrometry was used to quantify the four index compounds (wedelolactone, demethylwedelolactone, luteolin, apigenin) in these polyploid plants, and fluctuating patterns were detected. This is the first report regarding polyploidy in the herbal plant W. chinensis.

Transformation and Characterization of Δ12-Fatty Acid Acetylenase and Δ12-Oleate Desaturase Potentially Involved in the Polyacetylene Biosynthetic Pathway from Bidens pilosa.

Bidens pilosa is commonly used as an herbal tea component or traditional medicine for treating several diseases, including diabetes. Polyacetylenes have two or more carbon-carbon triple bonds or alkynyl functional groups and are mainly derived from fatty acid and polyketide precursors. Here, we report the cloning of full-length cDNAs that encode Δ12-fatty acid acetylenase (designated BPFAA) and Δ12-oleate desaturase (designated BPOD) from B. pilosa, which we predicted to play a role in the polyacetylene biosynthetic pathway. Subsequently, expression vectors carrying BPFAA or BPOD were constructed and transformed into B. pilosa via the Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of transgenes and selection marker genes in the obtained transgenic lines. The copy numbers of transgenes in transgenic lines were determined by Southern blot analysis. Furthermore, 4-5 FAA genes and 2-3 OD genes were detected in wild-type (WT) plants. Quantitative real time-PCR revealed that some transgenic lines had higher expression levels than WT. Western blot analysis revealed OD protein expression in the selected transformants. High-performance liquid chromatography profiling was used to analyze the seven index polyacetylenic compounds, and fluctuation patterns were found.

Cloning and characterization of leaf senescence up-regulated genes in sweet potato.

Genes that are expressed during leaf senescence in sweet potato (Ipomoea batatas, cv. Tainong 57) were identified by the isolation of cDNA fragments with the mRNA differential display method. Eight senescence-associated cDNA clones for mRNAs differentially expressed during leaf senescence were obtained and characterized. Northern blot analysis indicated that all these clones represented genes that are up-regulated during natural leaf senescence. Among them, five cDNA clones have been obtained in full length by screening a senescing leaf cDNA library or by performing rapid amplification of cDNA ends. DNA and protein database searches revealed that clones SPA15 and SPC9 encode proteins of unknown function. The other six clones SPG31, SPC20, SPG27, SPC25, SPC15 and SPC1 showed significant sequence homology to known genes encoding a cysteine proteinase, isocitrate lyase, S-adenosylmethionine decarboxylase, cysteine proteinase inhibitor and metallothionein-like type I protein. The gene expression patterns represented by SPG31, SPG27 and SPA15 were found to be highly specific in senescing leaves. The corresponding transcripts for SPG31, SPG27 and SPA15 were below detectable levels in other organs such as flowers, stems, roots and tubers. The possible physiological roles of these gene products in the leaf senescence process are discussed.