Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid–reversible net uptake of [³H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized ( S )-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli . The final yield of ( S )-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of (15)N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early- and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.

The secondary metabolites of higher plants include diverse chemicals, such as alkaloids, isoprenoids and phenolic compounds (phenylpropanoids and flavonoids). Although these compounds are widely used in human health and nutrition, at present they are mainly obtained by extraction from plants and extraction yields are low because most of these metabolites accumulate at low levels in plant cells. Recent advances in synthetic biology and metabolic engineering have enabled tailored production of plant secondary metabolites in microorganisms, but these methods often require the addition of expensive substrates. Here we develop an Escherichia coli fermentation system that yields plant alkaloids from simple carbon sources, using selected enzymes to construct a tailor-made biosynthetic pathway. In this system, engineered cells cultured in growth medium without additional substrates produce the plant benzylisoquinoline alkaloid, (S)-reticuline (yield, 46.0 mg l(-1) culture medium). The fermentation platform described here offers opportunities for low-cost production of many diverse alkaloids.

Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica , a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter ( Cjmdr1 ) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1 -injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome.

Combinations of mutations in miR319 and TCP transcription factor genes in Arabidopsis thaliana generate cotyledon fusion and various leaf forms with a broad ran