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

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

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

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

Mitochondrial ACONITASE3 is important for the acclimation to submergence stress by integrating carbon and nitrogen metabolism and impacting stress signaling pathways. 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.Peer reviewe

Proteome analysis of the Albugo candida–Brassica juncea pathosystem reveals that the timing of the expression of defence-related genes is a crucial determinant of pathogenesis

White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell's immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida–Brassica juncea pathosystem, especially in relation to host resistance to this pathogen

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

Magnetic quantitative reverse transcription PCR: A high-throughput method for mRNA extraction and quantitative reverse transcription PCR

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

Characterization of TCTP, the translationally controlled tumor protein, from Arabidopsis thaliana

The translationally controlled tumor protein (TCTP) is an important component of the TOR (target of rapamycin) signaling pathway, the major regulator of cell growth in animals and fungi. TCTP acts as the guanine nucleotide exchange factor of the Ras GTPase Rheb that controls TOR activity in Drosophila melanogaster. We therefore examined the role of Arabidopsis thaliana TCTP in planta. Plant TCTPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. Green fluorescent protein reporter lines show that Arabidopsis TCTP is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells. Knockout of TCTP leads to a male gametophytic phenotype with normal pollen formation and germination but impaired pollen tube growth. Silencing of TCTP by RNA interference slows vegetative growth; leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCTP as an important regulator of growth in plants and imply a function of plant TCTP as a mediator of TOR activity similar to that known in nonplant systems

Sulfur-Enhanced Defence: Effects of Sulfur Metabolism, Nitrogen Supply and Pathogen Lifestyle

Evidence from field experiments indicates differential roles of sulfur and nitrogen supply for plant resistance against pathogens. Dissection of these observations in defined pathosystems and controlled nutritional conditions indicates an activation of plant sulfur metabolism in several incompatible and compatible interactions. Contents of cysteine and glutathione as markers of primary sulfate assimilation and stress response show increases in Arabidopsis thaliana upon infection, coinciding with the synthesis of sulfur-containing defence compounds. Similar increases of thiols were observed with necrotrophic, biotrophic, and hemibiotrophic pathogens. Sulfate supply was found to be neutral or beneficial for tolerance against fungal but neutral for bacterial pathogens under in vitro conditions. According to various reports and own observations the effects of nitrogen supply appeared to be neutral or harmful, depending on the pathogen. The activation of sulfur metabolism was a consequence of activation of gene expression as revealed by macroarray analysis of an A. thaliana/Alternaria brassicicola pathosystem. This activation appeared to be largely independent from sufficient or optimal sulfate supply and from the established sulfate deficiency response. The data suggest that plant-pathogen interactions and sulfur metabolism are linked by jasmonic acid as signal