Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 – PHR2 complex

Phosphate (Pi) is a key macronutrient limiting plant growth and crop productivity. In response to the nutrient deficiency, Pi starvation response (PHR) transcription factors activate Pi starvation induced (PSI) genes. PHR transcription factors are negatively regulated by stand-alone SPX proteins, cellular receptors for inositol pyrophosphate (PP-InsP) nutrient messengers. How PP-InsP-bound SPX domains interact with PHR transcription factors is poorly understood. Here, we report crystal structures of the rice SPX2/InsP6/PHR2 complex and of the PHR2 DNA binding (MYB) domain in complex with its target DNA at resolutions of 3.1 Å and 2.7 Å, respectively. Inositol polyphosphate binding causes SPX2 to assemble into a domain-swapped dimer. The signalling-active SPX2 dimer binds two copies of PHR2, targeting both its coiled-coil (CC) oligomerisation domain and its MYB domain. Structural comparisons, biochemical analyses and genetic characterizations reveal that the SPX2 senses InsP6 / PP-InsPs to inactivate PHR2 by establishing severe steric clashes with the PHR2 MYB domain, preventing DNA binding, and by disrupting oligomerisation of the PHR2 CC domain, attenuating promoter binding. The complex structure rationalizes how PP-InsPs activate SPX receptor proteins to target PHR family transcription factors and provides a mechanistic framework to engineer crops with improved phosphate use efficiency.

Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1)

ABSTRACTIRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot mobilization of Fe. We show that the irt1-2 (pam42) mutant over-accumulates Fe in roots, most prominently in the cortex of the differentiation zone, when compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. In the transgenic Arabidopsis lines, short-term root Fe uptake rates and secondary substrate Mn accumulation resemble those of irt1-2, suggesting that these plants remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve iron nutrition and the nutritional quality of agricultural crops.

Comparative transcriptomic analysis reveals key components controlling spathe color in Anthurium andraeanum (Hort.)

ABSTRACTAnthurium andraeanum (Hort.) is an important ornamental in the tropical cut-flower industry. However, there is currently not enough information to establish a clear connection between the genetic model(s) proposed and the putative genes involved in the differentiation between colors. In this study, 18 cDNA libraries related to the spathe color and developmental stages of A. andraeanum cut-flowers were characterized by transcriptome sequencing technology. For the de novo transcriptome, a total of 114,334,082 primary sequence reads were obtained from the Illumina sequencer and were assembled into 151,652 unigenes. Approximately 58,476 transcripts were generated and used for comparative transcriptome analysis between three varieties that differ in spathe color (‘Sasha’ (white), ‘Honduras’ (red), and ‘Rapido’ (purple)). A large number of differentially expressed genes (8,324) that were potentially involved in multiple biological and metabolic pathways were identified, including the flavonoid and anthocyanin biosynthetic pathways. Our results showed that chalcone synthase (CHS) and flavonoid 3’-hydroxylase (F3’H) were the main genes differentially expressed in the white/red/purple comparison. We also identified a differentially expressed cytochrome P450 in the late developmental stage of the purple spathe that appeared to determine the difference between the red- and purple-colored spathes. Additionally, putative MYB-domain protein candidates that could be responsible for the control of the biosynthetic pathway were identified. The results provided basic sequence information for future research on spathe color, which have important implications for breeding strategies in this ornamental.Core ideasRNA-seq was performed on three anthurium varieties.Gene expression was compared for developmental stage and spathe color.Differentially expressed unigenes were identified.Putative MYB-domain protein candidates of the anthocyanin biosynthetic pathway were identified.

Identification of candidate genes controlling black seed coat and pod tip color in cowpea (Vigna unguiculata L. Walp)

ABSTRACTSeed coat color is an important part of consumer preferences for cowpea (Vigna unguiculata L. Walp). Color has been studied in numerous crop species and has often been linked to loci controlling the anthocyanin biosynthesis pathway. This study makes use of available resources, including mapping populations, a reference genome, and a high-density single nucleotide polymorphism genotyping platform, to map the black seed coat and purple pod tip color traits in cowpea. Several gene models encoding MYB domain protein 113 were identified as candidate genes. MYB domain proteins have been shown in other species to control expression of genes encoding enzymes for the final steps in the anthocyanin biosynthesis pathway. PCR analysis indicated that a presence/absence variation of one or more MYB113 genes may control the presence or absence of black pigment. A PCR marker has been developed for black seed coat color in cowpea.

The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock.Luxmutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein–DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using theMeshAndCollectworkflow available at the ESRF.