SciCombinator

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Journal: Plant physiology

169

Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.

Concepts: Pinophyta, Pine, Pinus classification, Resin, Lodgepole Pine, Conifer cone, Pinus, Abietic acid

169

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.

Concepts: Metabolism, Adenosine triphosphate, Enzyme, Starch, Tomato, Solanaceae, Solanum, Potato

167

Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by Artificial Neural Network Inference Analysis (ANN) and its function validated in transgenic plants. A tomato-fruit gene-regulatory network was generated using ANN and transcription-factor gene-expression profiles (Tfs) derived from fruits sampled at various points during development and ripening. One of the Tfs with a sequence related to an Arabidopsis PSEUDO RESPONSE REGULATOR 2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild type tomato fruits and, when over expressed in transgenic lines, increased plastid number, area and pigment content; enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines over expressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the over-expression lines providing a link between expression of this tomato gene and the ripening process. A putative orthologue of the tomato APPR2-Like gene in sweet pepper was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

Concepts: Gene expression, Fruit, Tomato, Solanaceae, Vegetable, Eggplant, Ethylene, Ripening

160

Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the Red light:Far Red light (R:FR) provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. As auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis thaliana deficient in phyB, but had a modest effect in WT. While indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared to WT. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than WT. Since the auxin response could be due either to the bud’s ability to export auxin into the main shoot PATS, or to auxin signaling status, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared to WT, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.

Concepts: Bud, Arabidopsis thaliana, Arabidopsis, Plant physiology, Plant stem, Shoot, Phytochrome, The Stems

160

Phytoplasmas have the smallest genome among bacteria and lack many essential genes required for biosynthetic and metabolic functions, making them unculturable, phloem-limited plant pathogens. In this study, we observed that transgenic Arabidopsis thaliana expressing the secreted effector protein SAP11AYWB of the Aster Yellows phytoplasma strain Witches' Broom (AY-WB) shows an altered root architecture, similarly to the disease symptoms of phytoplasma-infected plants, by forming hairy roots. This morphological change is paralleled by an accumulation of cellular Pi and an increase in the expression levels of Pi starvation-induced genes and miRNAs. In addition to the Pi starvation responses, we found that SAP11AYWB suppresses salicylic acid-mediated defense responses and enhances the growth of a bacterial pathogen. These results contribute to an improved understanding of the role of phytoplasma effector SAP11 and provide new insights for understanding the molecular basis of plant-pathogen interactions.

Concepts: DNA, Gene expression, Bacteria, Molecular biology, Metabolism, Genome, Phytoplasma, Plant pathogens and diseases

151

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca(2+) uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H(+))/K(+) symporter that mediates a high-affinity uptake during K(+) deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K(+) efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K(+) loss. Taken together, our results position ILK1 as a link between plant defense pathways and K(+) homeostasis.

Concepts: Immune system, Gene, Cell, Bacteria, Innate immune system, Pattern recognition receptor, Pathogen-associated molecular pattern, Osmosis

151

The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals, cytokinins and strigolactones, which can move into axillary buds. The other involves suppression of auxin transport/canalization from axillary buds into the main stem and is enhanced by a low sink for auxin in the stem. In this theory, the relative ability of buds and stem to transport auxin controls bud outgrowth. Here we evaluate whether auxin transport is required or regulated during bud outgrowth in pea (Pisum sativum). The profound, systemic and long-term effects of the auxin transport inhibitor N-1-naphthylphthalamic acid had very little inhibitory effect on bud outgrowth in strigolactone deficient mutants. Strigolactones can also inhibit bud outgrowth in N-1-naphthylphthalamic acid-treated shoots that have greatly diminished auxin transport. Moreover strigolactones can inhibit bud outgrowth despite a much diminished auxin supply in in vitro or decapitated plants. These findings demonstrate that auxin sink strength in the stem is not important for bud outgrowth in pea. Consistent with alternative mechanisms of auxin-regulation of systemic signals, enhanced auxin biosynthesis in Arabidopsis thaliana can suppress branching in yuc1D plants compared to wild-type plants, but has no effect on bud outgrowth in a strigolactone-deficient mutant background.

Concepts: Bud, Annual plant, Plant physiology, Plant hormone, Plant stem, Shoot, Auxin, Polar auxin transport

143

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape is the third most productive vegetable oil crop on the global market. Therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionisation - mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MADLI-MSI to study the spatial distribution of two major lipid species, triacylglycerols (TAGs) and phosphatidylcholines (PCs). A dramatic, heterogeneous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various seed tissues. The embryonic axis was particularly enriched in lipid species containing palmitate, while the seed coat/aleurone layer accumulated vaccenic, linoleic and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed to each other, a remarkable discovery given the supposed identical functionality of these two tissues. TAG and PC molecular species distribution was analysed through a developmental time series covering early seed lipid accumulation to the end of lipid accumulation. The spatial patterning of lipid molecular species did not vary much during seed development, although there were exceptions. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed implies differential regulation of lipid metabolism between the different seed tissues. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

Concepts: Mass spectrometry, Fat, Lipid, Rapeseed, Brassica, Palm oil, Vegetable fats and oils, Monoglyceride

132

The transition to flowering is a crucial step in the plant life cycle that is controlled by multiple endogenous and environmental cues, including hormones, sugars, temperature, and photoperiod. Permissive photoperiod induces the expression of FLOWERING LOCUS T (FT) in the phloem companion cells (PCC) of leaves. The FT protein then acts as a florigen that is transported to the shoot apical meristem (SAM), where it physically interacts with the bZIP transcription factor FD and 14-3-3 proteins. However, despite the importance of FD in promoting flowering, its direct transcriptional targets are largely unknown. Here, we combined ChIP-seq and RNA-seq to identify targets of FD at the genome scale and assessed the contribution of FT to DNA binding. We further investigated the ability of FD to form protein complexes with FT and TERMINAL FLOWER 1 (TFL1) through interaction with 14-3-3 proteins. Importantly, we observed direct binding of FD to targets involved in several aspects of plant development. These target genes were previously unknown to be directly related to the regulation of flowering time. Our results confirm FD as a central regulator of floral transition at the shoot meristem and provide evidence for crosstalk between the regulation of flowering and other signaling pathways, such as pathways involved in hormone signaling.

97

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a non-bitter sweet kernel. However, in the field there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine to mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. Here, this was accomplished by constructing a sequence database containing UGTs known, or prospected to catalyse a β(1→6)-O glycosylation reaction, and a BLAST search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana. Reverse transcription quantitative PCR demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two cytochrome P450s catalyzing the first steps in amygdalin biosynthesis.