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Plants use several pathways to synthesize phosphatidylcholine (PC), the major phospholipid of eukaryotic cells. PC has important structural and signaling roles. One pathway plants use for synthesis is the phospho-base methylation pathway, which forms the head-group phosphocholine through the triple methylation of phosphoethanolamine (PEA) catalyzed by phosphoethanolamine N-methyltransferases (PEAMTs). Our understanding of that pathway and its physiological importance remains limited. We recently reported that disruption of Arabidopsis thaliana PEAMT1/NMT1 and PEAMT3/NMT3 induces severe PC deficiency leading to dwarfism and impaired development. However, the double nmt1 nmt3 knock-out mutant is viable. Here, we show that this is enabled by residual PEAMT activity through a third family member, NMT2. The triple nmt1 nmt2 nmt3 knock-out mutant cannot synthesize PC from PEA and is lethal. This shows that, unlike mammals and yeast, Arabidopsis cannot form PC from phosphatidyl ethanolamine (PE), and demonstrates that methylation of PEA is the sole, and vital, entry point to PC synthesis. We further show that Arabidopsis has evolved an expanded family of four nonredundant PEAMTs through gene duplication and alternate use of the NMT2 promoter. NMT2 encodes two PEAMT variants, which greatly differ in their ability to perform the initial phospho-base methylation of PEA. Five amino acids at the N terminus of PEAMTs are shown to each be critical for the catalysis of that step committing to PC synthesis. As a whole, these findings open new avenues for enzymatic engineering and the exploration of ways to better tune phosphocholine and PC synthesis to environmental conditions for improved plant performance.

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin-regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

Interactions between phytohormones play important roles in the regulation of plant growth and development, but knowledge of the networks controlling hormonal relationships, such as between oxylipins and auxins, is just emerging. Here, we report the transc

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7irx3-5 involved in the control of TE. Leaves of the AtCesA7irx3-5 mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7irx3-5 mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7irx3-5 when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency.

Over the past few years high-throughput platforms for real-time quantitative PCR have become widely available. The cost of RNA extraction from a large number of samples are, however, quite notable. One method that stands out with respect to free up- or downscaling of sample size and reliability is the isolation of mRNA using oligodeoxythymidylate [oligo(dT)25]-coated magnetic particles. In combining this magnetic separation of mRNA with real-time reverse transcription PCR (RT-PCR), we have achieved a highly reproducible, economic, and fast way of analyzing large sample numbers. One difficulty that has so far prevented the fusion of these techniques relates to accurate mRNA quantification. We present a solution to this problem that enables excellent adjustment of cDNA amounts prior to the real-time PCR. Furthermore, as the mRNA is rapidly isolated from crude plant extracts, our method is widely applicable to herbaceous plant species and various tissue types without cumbersome adjustments. Although designed and tested here for plants, we anticipate that the principles should be applicable to gene expression studies in any other organism. Lastly, due to its flexibility, the method presented here can easily be adapted to specific requirements of various users and has great potential for further automation.

A transgenic approach was undertaken to investigate the role of a rice ammonium transporter (OsAMT1-1) in ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Transgenic lines overexpressing OsAMT1-1 were produced by Agrobacterium-mediated transformation of two rice cultivars, Taipei 309 and Jarrah, with an OsAMT1-1 cDNA gene construct driven by the maize ubiquitin promoter. Transcript levels of OsAMT1-1 in both Taipei 309 and Jarrah transgenic lines correlated positively with transgene copy number. Shoot and root biomass of some transgenic lines decreased during seedling and early vegetative stage compared to the wild type, especially when grown under high (2 mM) ammonium nutrition. Transgenic plants, particularly those of cv. Jarrah recovered in the mid-vegetative stage under high ammonium nutrition. Roots of the transgenic plants showed increased ammonium uptake and ammonium content. We conclude that the decreased biomass of the transgenic lines at early stages of growth might be caused by the accumulation of ammonium in the roots owing to the inability of ammonium assimilation to match the greater ammonium uptake.

The cause for the wave-like growth of Arabidopsis thaliana roots on semi-solid medium remains unclear. Researchers have hypothesized a gravity-induced touch-response, circumnutation, or combinations thereof act as the major stimuli. Our data demonstrate that the gaseous environment within the Petri dish can override gravitational effects. Furthermore, we show that medium ion concentrations and gelling polymers modify the wave response. Although the mechanisms driving our wide-ranging wild-type phenotypes are currently unknown, these results are of immediate significance for interpreting genetic and physiological modifications of environmentally and genetically induced characteristics.