Conservation of land plant-specific receptor-like cytoplasmic kinase subfamily XI possessing a unique kinase insert domain.

The number of genes encoding receptor-like kinases (RLKs) has expanded in the plant lineage. Their expansion has resulted in the emergence of diverse domain architectures that function in signaling cascades related to growth, development, and stress response. In this study, we focused on receptor-like cytoplasmic kinase subfamily XI (RLCK XI) in plants. We discovered an exceptionally long kinase insert domain (KID), averaging 280 amino acids, between subdomains VII and VIII of the conserved protein kinase domain. Using sequence homology search, we identified members of RLCK XI with the unique KID architecture in terrestrial plants, up to a single copy in several hornwort and liverwort species. The KID shows a high propensity for being disordered, resembling the activation segment in the model kinase domain. Several conserved sequence motifs were annotated along the length of the KID. Of note, the KID harbors repetitive nuclear localization signals capable of mediating RLCK XI translocation from the plasma membrane to the nucleus. The possible physiological implication of dual localization of RLCK XI members is discussed. The presence of a KID in RLCK XI represents a unique domain architecture among RLKs specific to land plants.

Phosphate transporter PHT1;1 is a key determinant of phosphorus acquisition in Arabidopsis natural accessions.

Phosphorus (P) is a mineral nutrient essential for plant growth and development, but most P in the soil is unavailable for plants. To understand the genetic basis of P acquisition regulation, we performed genome-wide association studies (GWASs) on a diversity panel of Arabidopsis (Arabidopsis thaliana). Two primary determinants of P acquisition were considered, namely, phosphate (Pi)-uptake activity and PHOSPHATE TRANSPORTER 1 (PHT1) protein abundance. Association mapping revealed a shared significant peak on chromosome 5 (Chr5) where the PHT1;1/2/3 genes reside, suggesting a connection between the regulation of Pi-uptake activity and PHT1 protein abundance. Genes encoding transcription factors, kinases, and a metalloprotease associated with both traits were also identified. Conditional GWAS followed by statistical analysis of genotype-dependent PHT1;1 expression and transcriptional activity assays revealed an epistatic interaction between PHT1;1 and MYB DOMAIN PROTEIN 52 (MYB52) on Chr1. Further, analyses of F1 hybrids generated by crossing two subgroups of natural accessions carrying specific PHT1;1- and MYB52-associated single nucleotide polymorphisms (SNPs) revealed strong effects of these variants on PHT1;1 expression and Pi uptake activity. Notably, the soil P contents in Arabidopsis habitats coincided with PHT1;1 haplotype, emphasizing how fine-tuned P acquisition activity through natural variants allows environmental adaptation. This study sheds light on the complex regulation of P acquisition and offers a framework to systematically assess the effectiveness of GWAS approaches in the study of quantitative traits.

Phosphate excess increases susceptibility to pathogen infection in rice.

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease.

Evolution of microRNA827 targeting in the plant kingdom.

Unlike most ancient microRNAs, which conservatively target homologous genes across species, microRNA827 (miR827) targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes, NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in Arabidopsis thaliana and Oryza sativa to regulate phosphate (Pi) transport and storage, respectively. However, how miR827 shifted its target preference and its evolutionary history are unknown. Based on target prediction analysis, we found that in most angiosperms, miR827 conservatively targets PHT5 homologs, but in Brassicaceae and Cleomaceae it preferentially targets NLA homologs, and we provide evidence for the transition of target preference during Brassicales evolution. Intriguingly, we found a lineage-specific loss of the miR827-regulatory module in legumes. Analysis of miR827-mediated cleavage efficiency and the expression of PHT5 in A. thaliana indicated that accumulation of mutations in the target site and the exclusion of the target site by alternative transcriptional initiation eliminated PHT5 targeting by miR827. Here, we identified a transition of miR827 target preference during plant evolution and revealed the uniqueness of miR827-mediated regulation among conserved plant miRNAs. Despite the change in its target preference, upregulation of miR827 by Pi starvation and its role in regulating cellular Pi homeostasis were retained.

Identification of plant vacuolar transporters mediating phosphate storage.

Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. Here we demonstrate that the SPX-MFS proteins, designated as PHOSPHATE TRANSPORTER 5 family (PHT5), also named Vacuolar Phosphate Transporter (VPT), function as vacuolar Pi transporters. Based on (31)P-magnetic resonance spectroscopy analysis, Arabidopsis pht5;1 loss-of-function mutants accumulate less Pi and exhibit a lower vacuolar-to-cytoplasmic Pi ratio than controls. Conversely, overexpression of PHT5 leads to massive Pi sequestration into vacuoles and altered regulation of Pi starvation-responsive genes. Furthermore, we show that heterologous expression of the rice homologue OsSPX-MFS1 mediates Pi influx to yeast vacuoles. Our findings show that a group of Pi transporters in vacuolar membranes regulate cytoplasmic Pi homeostasis and are required for fitness and plant growth.

Arabidopsis inositol pentakisphosphate 2-kinase, AtIPK1, is required for growth and modulates phosphate homeostasis at the transcriptional level.

Inositol hexakisphosphate (IP6 ) provides a phosphorous reservoir in plant seeds; in addition, along with its biosynthesis intermediates and derivatives, IP6 also plays important roles in diverse developmental and physiological processes. Disruption of the Arabidopsis inositol pentakisphosphate 2-kinase coding gene AtIPK1 was previously shown to reduce IP6 content in vegetative tissues and affect phosphate (Pi) sensing. Here we show that AtIPK1 is required for sustaining plant growth, as null mutants are non-viable. An incomplete loss-of-function mutant, atipk1-1, exhibited disturbed Pi homeostasis and overaccumulated Pi as a consequence of increased Pi uptake activity and root-to-shoot Pi translocation. The atipk1-1 mutants also showed a Pi deficiency-like root system architecture with reduced primary root and enhanced lateral root growth. Transcriptome analysis indicated that a subset of Pi starvation-responsive genes was transcriptionally perturbed in the atipk1-1 mutants and the expression of multiple genes involved in Pi uptake, allocation, and remobilization was increased. Genetic and transcriptional analyses suggest that disturbance of Pi homeostasis caused by atipk1 mutation involved components in addition to PHR1(-like) transcription factors. Notably, the transcriptional increase of a number of Pi starvation-responsive genes in the atipk1-1 mutants is correlated with the reduction of histone variant H2A.Z occupation in chromatin. The myo-inositol-1-phosphate synthase mutants, atmips1 and atmips2 with comparable reduction in vegetative IP6 to that in the atipk1-1 mutants did not overaccumulate Pi, suggesting that Pi homeostasis modulated by AtIPK1 is not solely attributable to IP6 level. This study reveals that AtIPK1 has important roles in growth and Pi homeostasis.

Regulatory network of microRNA399 and PHO2 by systemic signaling.

Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme 24. Arabidopsis (Arabidopsis thaliana) plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots. The association of Pi translocation and coexpression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. In this study, we used reciprocal grafting between wild-type and miR399-overexpressing transgenic plants to dissect the systemic roles of miR399 and PHO2. Arabidopsis rootstocks overexpressing miR399 showed high accumulation of Pi in the wild-type scions because of reduced PHO2 expression in the rootstocks. Although miR399 precursors or expression was not detected, we found a small but substantial amount of mature miR399 in the wild-type rootstocks grafted with transgenic scions, which indicates the movement of miR399 from shoots to roots. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also discovered in grafted tobacco (Nicotiana benthamiana) plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 small interfering RNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency is discussed.

Regulation of phosphate homeostasis by MicroRNA in Arabidopsis.

In this study, we reveal a mechanism by which plants regulate inorganic phosphate (Pi) homeostasis to adapt to environmental changes in Pi availability. This mechanism involves the suppression of a ubiquitin-conjugating E2 enzyme by a specific microRNA, miR399. Upon Pi starvation, the miR399 is upregulated and its target gene, a ubiquitin-conjugating E2 enzyme, is downregulated in Arabidopsis thaliana. Accumulation of the E2 transcript is suppressed in transgenic Arabidopsis overexpressing miR399. Transgenic plants accumulated five to six times the normal Pi level in shoots and displayed Pi toxicity symptoms that were phenocopied by a loss-of-function E2 mutant. Pi toxicity was caused by increased Pi uptake and by translocation of Pi from roots to shoots and retention of Pi in the shoots. Moreover, unlike wild-type plants, in which Pi in old leaves was readily retranslocated to other developing young tissues, remobilization of Pi in miR399-overexpressing plants was impaired. These results provide evidence that miRNA controls Pi homeostasis by regulating the expression of a component of the proteolysis machinery in plants.