Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

Plant defense responses are tightly controlled by many positive and negative regulators to cope with attacks from various pathogens. Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE2 (EDR2) is a negative regulator of powdery mildew resistance, and edr2 mutants display enhanced resistance to powdery mildew (Golovinomyces cichoracearum). To identify components acting in the EDR2 pathway, we screened for edr2 suppressors and identified a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes a calmodulin-binding transcription activator. The sr1-4D gain-of-function mutation suppresses all edr2-associated phenotypes, including powdery mildew resistance, mildew-induced cell death, and ethylene-induced senescence. The sr1-4D single mutant is more susceptible to a Pseudomonas syringae pv tomato DC3000 virulent strain and to avirulent strains carrying avrRpt2 or avrRPS4 than the wild type. We show that SR1 directly binds to the promoter region of NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a key component in RESISTANCE TO PSEU-DOMONAS SYRINGAE2-mediated plant immunity. Also, the ndr1 mutation suppresses the sr1-1 null allele, which shows enhanced resistance to both P. syringae pv tomato DC3000 avrRpt2 and G. cichoracearum. In addition, we show that SR1 regulates ethylene-induced senescence by directly binding to the ETHYLENE INSENSITIVE3 (EIN3) promoter region in vivo. Enhanced ethylene-induced senescence in sr1-1 is suppressed by ein3. Our data indicate that SR1 plays an important role in plant immunity and ethylene signaling by directly regulating NDR1 and EIN3.

Virtually nothing is known about the mechanisms and enzymes responsible for the glycosylation of arabinogalactan proteins (AGPs). The glycosyltransferase 37 family contains plant-specific enzymes, which suggests involvement in plantspecific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode ␣(1,2)fucosyltransferases (FUTs) for AGPs. Multiple lines of evidence support this hypothesis. First, overexpression of the two genes in tobacco BY2 cells, known to contain nonfucosylated AGPs, resulted in a staining of transgenic cells with eel lectin, which specifically binds to terminal ␣-linked fucose. Second, monosaccharide analysis by high pH anion exchange chromatography and electrospray ionization mass spectrometry indicated the presence of fucose in AGPs from transgenic cell lines but not in AGPs from wild type cells. Third, detergent extracts from microsomal membranes prepared from transgenic lines were able to fucosylate, in vitro, purified AGPs from BY2 wild type cells. Susceptibility of [ 14 C]fucosylated AGPs to ␣(1,2)fucosidase, and not to ␣(1,3/4)fucosidase, indicated that an ␣(1,2) linkage is formed. Furthermore, dearabinosylated AGPs were not substrate acceptors for these enzymes, indicating that arabinosyl residues represent the fucosylation sites on these molecules. Testing of several polysaccharides, oligosaccharides, and glycoproteins as potential substrate acceptors in the fucosyl transfer reactions indicated that the two enzymes are specific for AGPs but are not functionally redundant because they differentially fucosylate certain AGPs. AtFUT4 and AtFUT6 are the first enzymes to be characterized for AGP glycosylation and further our understanding of cell wall biosynthesis.

Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. In this study, we demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of the stem cell transcription factor Krüppel-like factor 5 (KLF5) and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced glycogen synthase kinase-3β (GSK3β)-mediated KLF5 protein phosphorylation and degradation through the inhibition of protein kinase A (PKA) activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB, p-GSK3β, KLF5 and FGF-BP1 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3β-KLF5 signaling pathway.