Is N-Hydroxytryptamine an Intermediate in Auxin Biosynthesis?
Four biosynthetic pathways have been proposed for Trp-dependent indole-3-acetic acid (IAA) biosynthesis in plants: the indole-3-acetaldoxime, indole-3-pyruvic acid, indole-3-acetamide, and tryptamine pathways. None of these pathways has been fully characterized, although there have been significant advances in the past few years because of the identification of new auxin mutants and improved analytical techniques. Recently, the tryptamine pathway has received particular attention because of the identification of YUC genes. Previous studies, based largely on mass spectrometry or HPLC analyses, led to the suggestion that the YUC1 of Arabidopsis (Arabidopsis thaliana; AtYUC1) and its putative homologs in other species catalyze the formation of N-hydroxytryptamine from tryptamine. Tivendale et al. (pp. 1957–1965) now challenge several technical aspects of these previous studies. In this article, the authors present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the previously published mass spectral data are not consistent with N-hydroxytryptamine. The authors also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. These new findings lead to doubts concerning whether N-hydroxytryptamine serves as an intermediate for IAA biosynthesis as previously proposed.
Expression Patterns of a Highly Duplicated Gene Involved in Exocytosis
Exocytosis involving the fusion of Golgi-derived vesicles with the plasma membrane is an essential process for plant growth and development. During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. The evolutionary conserved exocyst complex consists of eight subunits, one of which is EXO70. In contrast to a single copy of the EXO70 gene in fungal and most animal genomes, plants have a multitude of EXO70 genes: 13 in Physcomitrella patens, 23 in Arabidopsis and Populus trichocarpa, and 41 in rice (Oryza sativa), with functions very much unknown. Li et al. (pp. 1819–1830) have sought to obtain a complete expression profile for all 23 EXO70 genes in the Arabidopsis genome. Reverse transcription-PCR was performed on all 23 EXO70 genes in Arabidopsis to examine their expression in organ levels. Cell-level expression analyses were performed using transgenic plants carrying GUS reporter constructs. The results show that EXO70 genes are primarily expressed in exocytosis-active cells, such as tip-growing and elongating cells, developing xylem elements, and guard cells. No expression was observed in cells of mature organs, such as well-developed leaves, stems, sepals, and petals. These findings imply that the multiplicity of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis.
Characterization of a Peroxisome Protein Import Mutant
Peroxisome proteins are synthesized on free ribosomes in the cytosol and imported posttranslationally. Peroxisome proteins contain one of two well-characterized peroxisomal targeting signals (PTS1 and PTS2) that direct target proteins to the peroxisome matrix. Approximately 400 genes in the Arabidopsis genome code for proteins with either PTS1 or PTS2 signals. A majority of these proteins contain PTS1, with only one-quarter having the PTS2 sequence. Proteins that play a role in peroxisome biogenesis are referred to as PEROXINS (PEX). PEX5 and PEX7 function as receptors that recognize PTS1 and PTS2 proteins, respectively, for matrix protein import. To better understand the role of PEX5 in plant peroxisomal import, Khan and Zolman (pp. 1602–1615) have characterized the pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins and enzymatic processes that occur in peroxisomes are disrupted. The pex5-10 mutant has germination defects and is completely dependent on exogenous sucrose for early seedling establishment, based on poor utilization of seed-storage fatty acids (FAs). Although adult pex5-10 plants appear normal, the mutant also has delayed development and reduced fertility. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate is disrupted also. To study the import of and importance of PTS1 proteins, the authors created a truncated PEX5 construct lacking the PTS1 binding region (PEX5454). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5454) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 with mutant transgenic lines expressing the truncated PEX5454 protein allowed the authors to separate the effects of defects in PTS1 and PTS2, alone or in combination. For example, an inability of the PEX5454 transgenic line to metabolize either propionate or isobutyrate results in a hypersensitive phenotype, suggesting that the enzymes (or at least the key rate-limiting enzymes) involved in processing these metabolites employ a PTS1 signal.
Metabolic Engineering of Seed ω-7 FA Accumulation
The global consumption of 1-octene is over half a million tons per year, and most of it is used as a comonomer in the expanding production of linear low-density polyethylene. 1-Octene is currently synthesized mainly from petroleum-derived ethylene. Plant oils containing ω-7 FAs (palmitoleic 16:1Δ9 and cis-vaccenic 18:1Δ11) harbor considerable potential as alternative sources for 1-octene. Several natural plant oil sources of ω-7 FA have been reported, e.g. milkweed (Asclepias syriaca) accumulates approximately 25% ω-7 FA and cat's claw vine (Doxantha unguis-cati) accumulates approximately 72% ω-7 FA, but the low yields and poor agronomic properties of these plants preclude their commercial use. Isolation of the Δ9-16:0-ACP desaturase genes responsible for the production of ω-7 FA from milkweed and Doxantha presented an opportunity for their heterologous expression and ω-7 FA production in transgenic crops. However, heterologous expression of the milkweed desaturase in Arabidopsis failed to produce detectable ω-7 FA, and when the Doxantha desaturase was expressed in Brassica napus, it resulted in the accumulation of only approximately 9% ω-7 FA. Nguyen et al. (pp. 1897–1904) have achieved high levels of ω-7 FA accumulation by systematic metabolic engineering of Arabidopsis. A plastidial 16:0-ACP desaturase was engineered to convert 16:0 to 16:1Δ9 with specificity >100-fold that of naturally occurring paralogs. Expressing this engineered enzyme (Com25) in seeds increased ω-7 FA accumulation from <2% to 14%. Reducing competition for the substrates needed by 16:0-ACP by down-regulating another enzyme (β-ketoacyl-ACP synthase II 16:0 elongase) further increased accumulation of ω-7 FA to 56%. Finally, coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level from 21% to 11% and increased ω-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.
Phloem Loading of S-Methyl-Met
Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methyl-Met (SMM), a derivative of Met, has been proposed to be an important long-distance transport form of reduced S in legumes and other plant species. It is hypothesized that SMM is synthesized from Met in source leaves and loaded into the phloem for transport to seeds. In the seed coat, SMM is recycled back to Met that is then taken up by the embryo/cotyledons for storage and development. Tan et al. (pp. 1886–1896) have examined the importance of phloem loading and the source-sink partitioning of SMM for plant metabolism and growth. Because there are no known plant SMM transporters, a high-affinity yeast SMM transporter, S-Methylmethionine Permease1 (MMP1), was expressed in the pea phloem and seeds to determine whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea plants. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. It was also established that the increased phloem loading of SMM affects S metabolism in leaves and S uptake and assimilation in roots. In addition, nitrogen (N) assimilation and translocation to sinks were altered in MMP1 plants. Changes in S and N metabolism and partitioning resulted in higher seed numbers and influenced the S-N ratio and storage protein profile in MMP1 pea seeds. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport and seed set.
Light Control of Chromatin Compaction
Light plays a crucial role in numerous plant developmental processes. As a consequence, variation in light conditions has an enormous impact on the life cycle of a plant. To deal with light intensity, spectral quality, light direction, and photoperiod, plants have developed signaling mechanisms that are based on light-sensitive receptors. Phytochromes (phyA–phyE) mainly mediate in the perception of red and far-red light, whereas cryptochromes (CRY1, CRY2) and phototropins (PHOT1, PHOT2) are involved in the perception of blue light and UV-A. Corresponding to their nuclear localization, cryptochromes and phytochromes are involved in the control of gene transcription. Specifically, these photoreceptors mediate light-stimulated degradation and stability of several transcription factors, leading to modifications in gene expression profiles. CRY2 is associated with chromatin, and it physically interacts with a transcription factor. It has been demonstrated previously that the large-scale reduction of chromatin compaction that occurs during floral transition is affected in the cry2 mutant. Moreover, it was recently demonstrated that phyb mutants display reduced chromatin compaction under standard light conditions. Together, these data indicate that light controls large-scale chromatin organization. These findings prompted van Zanten et al. (pp. 1686–1696) to address the question of how light signaling leads to changes in chromatin compaction. They report that a decrease in light intensity induces a large-scale reduction in chromatin compaction. This low light response is reversible and shows strong natural genetic variation. Moreover, the degree of chromatin compaction is affected by light quality signals relevant for natural canopy shade. CRY2 appears a general positive regulator of low light-induced chromatin decompaction. PhyB also controls light-induced chromatin organization, but its effect appears to be dependent on the genetic background (it was found in Arabidopsis Columbia-0 lines only). The authors propose that chromatin compaction is regulated by the light environment using CRY2 protein abundance, which, in turn, is controlled by phyB action.