Dynamic interactions between SPX proteins, the ubiquitination machinery, and signalling molecules for stress adaptation at a whole-plant level.

The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF-CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.

GWAS on multiple traits identifies mitochondrial ACONITASE3 as important for acclimation to submergence stress.

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions.

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the "stress tolerant" allele. The former allele's inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

SPX4 Acts on PHR1-Dependent and -Independent Regulation of Shoot Phosphorus Status in Arabidopsis.

Phosphorus (P) is an essential macronutrient for all living organisms and limits plant growth. Four proteins comprising a single SYG1/Pho81/XPR1 (SPX) domain, SPX1 to SPX4, are putative phosphate-dependent inhibitors of Arabidopsis (Arabidopsis thaliana) PHOSPHATE STARVATION RESPONSE1 (PHR1), the master transcriptional activator of phosphate starvation responses. This work demonstrated that SPX4 functions as a negative regulator not only of PHR1-dependent but also of PHR1-independent responses in P-replete plants. Transcriptomes of P-limited spx4 revealed that, unlike SPX1 and SPX2, SPX4 modulates the shoot phosphate starvation response but not short-term recovery after phosphate resupply. In roots, transcriptional regulation of P status is SPX4 independent. Genes misregulated in spx4 shoots intersect with both PHR1-dependent and PHOSPHATE2-dependent signaling networks associated with plant development, senescence, and ion/metabolite transport. Gene regulatory network analyses suggested that SPX4 interacts with transcription factors other than PHR1, such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN55, known regulators of shoot development. Transient expression studies in protoplasts indicated that PHR1 retention in the cytosol by SPX4 occurs in a dose- and P-status-dependent manner. Using a luciferase reporter in vivo, SPX4 expression kinetics and stability revealed that SPX4 is a short-lived protein with P-status-dependent turnover. SPX4 protein levels were quickly restored by phosphate resupply to P-limited plants. Unlike its monocot ortholog, AtSPX4 was not stabilized by the phosphate analog phosphite, implying that intracellular P status is sensed by its SPX domain via phosphate-rich metabolite signals.

NMT1 and NMT3 N-Methyltransferase Activity Is Critical to Lipid Homeostasis, Morphogenesis, and Reproduction.

Phosphatidylcholine (PC) is a major membrane phospholipid and a precursor for major signaling molecules. Understanding its synthesis is important for improving plant growth, nutritional value, and resistance to stress. PC synthesis is complex, involving several interconnected pathways, one of which proceeds from serine-derived phosphoethanolamine to form phosphocholine through three sequential phospho-base methylations catalyzed by phosphoethanolamine N-methyltransferases (PEAMTs). The contribution of this pathway to the production of PC and plant growth has been a matter of some debate. Although a handful of individual PEAMTs have been described, there has not been any in planta investigation of a PEAMT family. Here, we provide a comparative functional analysis of two Arabidopsis (Arabidopsis thaliana) PEAMTs, NMT1 and the little known NMT3. Analysis of loss-of-function mutants demonstrates that NMT1 and NMT3 synergistically regulate PC homeostasis, phase transition at the shoot apex, coordinated organ development, and fertility through overlapping but also specific functions. The nmt1 nmt3 double mutant shows extensive sterility, drastically reduced PC concentrations, and altered lipid profiles. These findings demonstrate that the phospho-base methylation pathway makes a major contribution to PC synthesis in Arabidopsis and that NMT1 and NMT3 play major roles in its catalysis and the regulation of PC homeostasis as well as in plant growth and reproduction.

Tight control of sulfur assimilation: an adaptive mechanism for a plant from a severely phosphorus-impoverished habitat.

Hakea prostrata (Proteaceae) has evolved in extremely phosphorus (P)-impoverished habitats. Unlike species that evolved in P-richer environments, it tightly controls its nitrogen (N) acquisition, matching its low protein concentration, and thus limiting its P requirement for ribosomal RNA (rRNA). Protein is a major sink for sulfur (S), but the link between low protein concentrations and S metabolism in H. prostrata is unknown, although this is pivotal for understanding this species' supreme adaptation to P-impoverished soils. Plants were grown at different sulfate supplies for 5 wk and used for nutrient and metabolite analyses. Total S content in H. prostrata was unchanged with increasing S supply, in sharp contrast with species that typically evolved in environments where P is not a major limiting nutrient. Unlike H. prostrata, other plants typically store excess available sulfate in vacuoles. Like other species, S-starved H. prostrata accumulated arginine, lysine and O-acetylserine, indicating S deficiency. Hakea prostrata tightly controls its S acquisition to match its low protein concentration and low demand for rRNA, and thus P, the largest organic P pool in leaves. We conclude that the tight control of S acquisition, like that of N, helps H. prostrata to survive in P-impoverished environments.

Root Cell-Specific Regulators of Phosphate-Dependent Growth.

Cellular specialization in abiotic stress responses is an important regulatory feature driving plant acclimation. Our in silico approach of iterative coexpression, interaction, and enrichment analyses predicted root cell-specific regulators of phosphate starvation response networks in Arabidopsis (Arabidopsis thaliana). This included three uncharacterized genes termed Phosphate starvation-induced gene interacting Root Cell Enriched (PRCE1, PRCE2, and PRCE3). Root cell-specific enrichment of 12 candidates was confirmed in promoter-GFP lines. T-DNA insertion lines of 11 genes showed changes in phosphate status and growth responses to phosphate availability compared with the wild type. Some mutants (cbl1, cipk2, prce3, and wdd1) displayed strong biomass gain irrespective of phosphate supply, while others (cipk14, mfs1, prce1, prce2, and s6k2) were able to sustain growth under low phosphate supply better than the wild type. Notably, root or shoot phosphate accumulation did not strictly correlate with organ growth. Mutant response patterns markedly differed from those of master regulators of phosphate homeostasis, PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHOSPHATE2 (PHO2), demonstrating that negative growth responses in the latter can be overcome when cell-specific regulators are targeted. RNA sequencing analysis highlighted the transcriptomic plasticity in these mutants and revealed PHR1-dependent and -independent regulatory circuits with gene coexpression profiles that were highly correlated to the quantified physiological traits. The results demonstrate how in silico prediction of cell-specific, stress-responsive genes uncovers key regulators and how their manipulation can have positive impacts on plant growth under abiotic stress.

The loss of vernalization requirement in narrow-leafed lupin is associated with a deletion in the promoter and de-repressed expression of a Flowering Locus T (FT) homologue.

Adaptation of Lupinus angustifolius (narrow-leafed lupin) to cropping in southern Australian and northern Europe was transformed by a dominant mutation (Ku) that removed vernalization requirement for flowering. The Ku mutation is now widely used in lupin breeding to confer early flowering and maturity. We report here the identity of the Ku mutation. We used a range of genetic, genomic and gene expression approaches to determine whether Flowering Locus T (FT) homologues are associated with the Ku locus. One of four FT homologues present in the narrow-leafed lupin genome, LanFTc1, perfectly co-segregated with the Ku locus in a reference mapping population. Expression of LanFTc1 in the ku (late-flowering) parent was strongly induced by vernalization, in contrast to the Ku (early-flowering) parent, which showed constitutively high LanFTc1 expression. Co-segregation of this expression phenotype with the LanFTc1 genotype indicated that the Ku mutation impairs cis-regulation of LanFTc1. Sequencing of LanFTc1 revealed a 1.4-kb deletion in the promoter region, which was perfectly predictive of vernalization response in 216 wild and domesticated accessions. Linkage disequilibrium rapidly decayed around LanFTc1, suggesting that this deletion caused the loss of vernalization response. This is the first time a legume FTc subclade gene has been implicated in the vernalization response.

Tight control of nitrate acquisition in a plant species that evolved in an extremely phosphorus-impoverished environment.

Hakea prostrata (Proteaceae) has evolved in an extremely phosphorus (P)-limited environment. This species exhibits an exceptionally low ribosomal RNA (rRNA) and low protein and nitrogen (N) concentration in its leaves. Little is known about the N requirement of this species and its link to P metabolism, despite this being the key to understanding how it functions with a minimal P budget. H. prostrata plants were grown with various N supplies. Metabolite and elemental analyses were performed to determine its N requirement. H. prostrata maintained its organ N content and concentration at a set point, independent of a 25-fold difference nitrate supplies. This is in sharp contrast to plants that are typically studied, which take up and store excess nitrate. Plants grown without nitrate had lower leaf chlorophyll and carotenoid concentrations, indicating N deficiency. However, H. prostrata plants at low or high nitrate availability had the same photosynthetic pigment levels and hence were not physiologically compromised by the treatments. The tight control of nitrate acquisition in H. prostrata retains protein at a very low level, which results in a low demand for rRNA and P. We surmise that the constrained nitrate acquisition is an adaptation to severely P-impoverished soils.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.

Sensitivity of jarrah (Eucalyptus marginata) to phosphate, phosphite, and arsenate pulses as influenced by fungal symbiotic associations.

Many plant species adapted to P-impoverished soils, including jarrah (Eucalyptus marginata), develop toxicity symptoms when exposed to high doses of phosphate (Pi) and its analogs such as phosphite (Phi) and arsenate (AsV). The present study was undertaken to investigate the effects of fungal symbionts Scutellospora calospora, Scleroderma sp., and Austroboletus occidentalis on the response of jarrah to highly toxic pulses (1.5 mmol kg(-1) soil) of Pi, Phi, and AsV. S. calospora formed an arbuscular mycorrhizal (AM) symbiosis while both Scleroderma sp. and A. occidentalis established a non-colonizing symbiosis with jarrah plants. All these interactions significantly improved jarrah growth and Pi uptake under P-limiting conditions. The AM fungal colonization naturally declines in AM-eucalypt symbioses after 2-3 months; however, in the present study, the high Pi pulse inhibited the decline of AM fungal colonization in jarrah. Four weeks after exposure to the Pi pulse, plants inoculated with S. calospora had significantly lower toxicity symptoms compared to non-mycorrhizal (NM) plants, and all fungal treatments induced tolerance against Phi toxicity in jarrah. However, no tolerance was observed for AsV-treated plants even though all inoculated plants had significantly lower shoot As concentrations than the NM plants. The transcript profile of five jarrah high-affinity phosphate transporter (PHT1 family) genes in roots was not altered in response to any of the fungal species tested. Interestingly, plants exposed to high Pi supplies for 1 day did not have reduced transcript levels for any of the five PHT1 genes in roots, and transcript abundance of four PHT1 genes actually increased. It is therefore suggested that jarrah, and perhaps other P-sensitive perennial species, respond positively to Pi available in the soil solution through increasing rather than decreasing the expression of selected PHT1 genes. Furthermore, Scleroderma sp. can be considered as a fungus with dual functional capacity capable of forming both ectomycorrhizal and non-colonizing associations, where both pathways are always accompanied by evident growth and nutritional benefits.

Differentiating phosphate-dependent and phosphate-independent systemic phosphate-starvation response networks in Arabidopsis thaliana through the application of phosphite.

Phosphite is a less oxidized form of phosphorus than phosphate. Phosphite is considered to be taken up by the plant through phosphate transporters. It can mimic phosphate to some extent, but it is not metabolized into organophosphates. Phosphite could therefore interfere with phosphorus signalling networks. Typical physiological and transcriptional responses to low phosphate availability were investigated and the short-term kinetics of their reversion by phosphite, compared with phosphate, were determined in both roots and shoots of Arabidopsis thaliana. Phosphite treatment resulted in a strong growth arrest. It mimicked phosphate in causing a reduction in leaf anthocyanins and in the expression of a subset of the phosphate-starvation-responsive genes. However, the kinetics of the response were slower than for phosphate, which may be due to discrimination against phosphite by phosphate transporters PHT1;8 and PHT1;9 causing delayed shoot accumulation of phosphite. Transcripts encoding PHT1;7, lipid-remodelling enzymes such as SQD2, and phosphocholine-producing NMT3 were highly responsive to phosphite, suggesting their regulation by a direct phosphate-sensing network. Genes encoding components associated with the 'PHO regulon' in plants, such as At4, IPS1, and PHO1;H1, generally responded more slowly to phosphite than to phosphate, except for SPX1 in roots and MIR399d in shoots. Two uncharacterized phosphate-responsive E3 ligase genes, PUB35 and C3HC4, were also highly phosphite responsive. These results show that phosphite is a valuable tool to identify network components directly responsive to phosphate.

Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate.

BACKGROUND: In plants, the uptake from soil and intercellular transport of inorganic phosphate (Pi) is mediated by the PHT1 family of membrane-spanning proton : Pi symporters. The Arabidopsis thaliana AtPHT1 gene family comprises nine putative high-affinity Pi transporters. While AtPHT1;1 to AtPHT1;4 are involved in Pi acquisition from the rhizosphere, the role of the remaining transporters is less clear. RESULTS: Pi uptake and tissue accumulation studies in AtPHT1;8 and AtPHT1;9 knock-out mutants compared to wild-type plants showed that both transporters are involved in the translocation of Pi from the root to the shoot. Upon inactivation of AtPHT1;9, changes in the transcript profiles of several genes that respond to plant phosphorus (P) status indicated a possible role in the regulation of systemic signaling of P status within the plant. Potential genetic interactions were found among PHT1 transporters, as the transcript profile of AtPHT1;5 and AtPHT1;7 was altered in the absence of AtPHT1;8, and the transcript profile of AtPHT1;7 was altered in the Atpht1;9 mutant. These results indicate that AtPHT1;8 and AtPHT1;9 translocate Pi from the root to the shoot, but not from the soil solution into the root. CONCLUSION: AtPHT1;8 and AtPHT1;9 are likely to act sequentially in the interior of the plant during the root-to-shoot translocation of Pi, and play a more complex role in the acclimation of A. thaliana to changes in Pi supply than was previously thought.

Lipid biosynthesis and protein concentration respond uniquely to phosphate supply during leaf development in highly phosphorus-efficient Hakea prostrata.

Hakea prostrata (Proteaceae) is adapted to severely phosphorus-impoverished soils and extensively replaces phospholipids during leaf development. We investigated how polar lipid profiles change during leaf development and in response to external phosphate supply. Leaf size was unaffected by a moderate increase in phosphate supply. However, leaf protein concentration increased by more than 2-fold in young and mature leaves, indicating that phosphate stimulates protein synthesis. Orthologs of known lipid-remodeling genes in Arabidopsis (Arabidopsis thaliana) were identified in the H. prostrata transcriptome. Their transcript profiles in young and mature leaves were analyzed in response to phosphate supply alongside changes in polar lipid fractions. In young leaves of phosphate-limited plants, phosphatidylcholine/phosphatidylethanolamine and associated transcript levels were higher, while phosphatidylglycerol and sulfolipid levels were lower than in mature leaves, consistent with low photosynthetic rates and delayed chloroplast development. Phosphate reduced galactolipid and increased phospholipid concentrations in mature leaves, with concomitant changes in the expression of only four H. prostrata genes, GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE1, N-METHYLTRANSFERASE2, NONSPECIFIC PHOSPHOLIPASE C4, and MONOGALACTOSYLDIACYLGLYCEROL3. Remarkably, phosphatidylglycerol levels decreased with increasing phosphate supply and were associated with lower photosynthetic rates. Levels of polar lipids with highly unsaturated 32:x (x = number of double bonds in hydrocarbon chain) and 34:x acyl chains increased. We conclude that a regulatory network with a small number of central hubs underpins extensive phospholipid replacement during leaf development in H. prostrata. This hard-wired regulatory framework allows increased photosynthetic phosphorus use efficiency and growth in a low-phosphate environment. This may have rendered H. prostrata lipid metabolism unable to adjust to higher internal phosphate concentrations.

Root architecture responses: in search of phosphate.

Soil phosphate represents the only source of phosphorus for plants and, consequently, is its entry into the trophic chain. This major component of nucleic acids, phospholipids, and energy currency of the cell (ATP) can limit plant growth because of its low mobility in soil. As a result, root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant, a strategy described as topsoil foraging. We will review the diverse developmental strategies that can be observed among plants by detailing the effect of phosphate deficiency on primary and lateral roots. We also discuss the formation of cluster roots: an advanced adaptive strategy to cope with low phosphate availability observed in a limited number of species. Finally, we will put this work into perspective for future research directions.

Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of Proteaceae species.

Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus ('delayed greening'), with young leaves having very low levels of chlorophyll and Calvin-Benson cycle enzymes. In mature leaves, soluble protein and Calvin-Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin-Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.

Organ-specific phosphorus-allocation patterns and transcript profiles linked to phosphorus efficiency in two contrasting wheat genotypes.

Recent studies have identified genotypic variation in phosphorus (P) efficiency, but rarely have the underlying mechanisms been described at the molecular level. We demonstrate that the highly P-efficient wheat (Triticum aestivum L.) cultivar Chinese 80-55 maintains higher inorganic phosphate (Pi ) concentrations in all organs upon Pi withdrawal in combination with higher Pi acquisition in the presence of Pi when compared with the less-efficient cultivar Machete. These findings correlated with differential organ-specific expression of Pi transporters TaPHT1;2, TaPHT1;5, TaPHT1;8, TaPHT2;1 and H(+) -ATPase TaHa1. Observed transcript level differences between the cultivars suggest that higher de novo phospholipid biosynthetic activities in Pi -limited elongating basal leaf sections are another crucial adaptation in Chinese 80-55 for sustaining growth upon Pi withdrawal. These activities may be supported through enhanced breakdown of starch in Chinese 80-55 stems as suggested by higher TaGPho1 transcript levels. Chinese 80-55 fine roots on the other hand show strong suppression of transcripts involved in glycolysis, transcriptional regulation and ribosomal activities. Our work reveals major differences in the way the two contrasting cultivars allocate Pi and organic P compounds between source and sink tissues and in the acclimation of their metabolism to changes in Pi availability.

A novel plant-fungus symbiosis benefits the host without forming mycorrhizal structures.

• Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. • Here, we combined nutritional, radioisotopic ((33)P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. • We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated (33)Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. • This work is a starting point for detailed mechanistic studies on other basidiomycete-woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.