Phosphate deficiency alters transcript isoforms via alternative transcription start sites.

Alternative transcription start sites (TSS) are widespread in eukaryotes and can alter the 5' UTR length and coding potential of transcripts. Here we show that inorganic phosphate (Pi) availability regulates the usage of several alternative TSS in Arabidopsis (Arabidopsis thaliana). In comparison to phytohormone treatment, Pi had a pronounced and specific effect on the usage of many alternative TSS. By combining short-read RNA sequencing with long-read sequencing of full-length mRNAs, we identified a set of 45 genes showing alternative TSS under Pi deficiency. Alternative TSS affected several processes, such as translation via the exclusion of upstream open reading frames present in the 5' UTR of RETICULAN LIKE PROTEIN B1 mRNA, and subcellular localization via removal of the plastid transit peptide coding region from the mRNAs of HEME OXYGENASE 1 and SULFOQUINOVOSYLDIACYLGLYCEROL 2. Several alternative TSS also generated shorter transcripts lacking the coding potential for important domains. For example, the EVOLUTIONARILY CONSERVED C-TERMINAL REGION 4 (ECT4) locus, which encodes an N6-methyladenosine (m6A) reader, strongly expressed under Pi deficiency a short noncoding transcript (named ALTECT4) ~550 nt long with a TSS in the penultimate intron. The specific and robust induction of ALTECT4 production by Pi deficiency led to the identification of a role for m6A readers in primary root growth in response to low phosphate that is dependent on iron and is involved in modulating cell division in the root meristem. Our results identify alternative TSS usage as an important process in the plant response to Pi deficiency.

Recent advances in unraveling the mystery of combined nutrient stress in plants.

Efficiently regulating growth to adapt to varying resource availability is crucial for organisms, including plants. In particular, the acquisition of essential nutrients is vital for plant development, as a shortage of just one nutrient can significantly decrease crop yield. However, plants constantly experience fluctuations in the presence of multiple essential mineral nutrients, leading to combined nutrient stress conditions. Unfortunately, our understanding of how plants perceive and respond to these multiple stresses remains limited. Unlocking this mystery could provide valuable insights and help enhance plant nutrition strategies. This review focuses specifically on the regulation of phosphorous homeostasis in plants, with a primary emphasis on recent studies that have shed light on the intricate interactions between phosphorous and other essential elements, such as nitrogen, iron, and zinc, as well as non-essential elements like aluminum and sodium. By summarizing and consolidating these findings, this review aims to contribute to a better understanding of how plants respond to and cope with combined nutrient stress.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

PHO1 family members transport phosphate from infected nodule cells to bacteroids in Medicago truncatula.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

A nuclear factor Y interacting protein of the GRAS family is required for nodule organogenesis, infection thread progression, and lateral root growth.

A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NF-YC1-interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes Cyclin B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.

A CYBDOM protein impacts iron homeostasis and primary root growth under phosphate deficiency in Arabidopsis.

Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants.

Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues.

Tritiated thymidine autoradiography, 5-bromo-2'-deoxyuridine (BrdU) 5-chloro-2'-deoxyuridine (CldU), 5-iodo-2'-deoxyuridine (IdU), and 5-ethynyl-2'-deoxyiridine (EdU) labeling have been used for identifying the fraction of cells undergoing the S-phase of the cell cycle and to follow the fate of these cells during the embryonic, perinatal, and adult life in several species of vertebrate. In this current review, I will discuss the dosage and times of exposition to the aforementioned thymidine analogues to label most of the cells undergoing the S-phase of the cell cycle. I will also show how to infer, in an asynchronous cell population, the duration of the G1, S, and G2 phases, as well as the growth fraction and the span of the whole cell cycle on the base of some labeling schemes involving a single administration, continuous nucleotide analogue delivery, and double labeling with two thymidine analogues. In this context, the choice of the optimal dose of BrdU, CldU, IdU, and EdU to label S-phase cells is a pivotal aspect to produce neither cytotoxic effects nor alter cell cycle progression. I hope that the information presented in this review can be of use as a reference for researchers involved in the genesis of tissues and organs.

Phosphate acquisition and metabolism in plants.

Plants need at least 13 different nutrients to maintain optimal growth. Nitrogen and phosphorus, from the Greek 'phôs' (meaning 'light') and 'phoros' (meaning 'bearer'), are the main nutrients limiting plant growth in both agricultural and natural ecosystems. Agriculture has relied heavily since the mid 1950s on the use of synthetic ammonium- and phosphorus-based fertilizers to increase crop productivity. While industrial synthesis of ammonium relies on the chemical conversion of atmospheric nitrogen, phosphorus is mined from finite reserves concentrated in a few countries. Considering our current dependence on phosphorus fertilizers for food production and the geopolitical aspects associated with current resources, it will be important to develop technologies enabling the maintenance of high crop yield with reduced fertilizer input. This will require an in-depth knowledge on the various pathways that enable plants to acquire phosphorus from the soil and maximize its economical use for growth and reproduction. In this primer, we give an overview of the factors limiting phosphorus acquisition by plants and highlight various pathways and strategies plants have evolved at the level of development, metabolism and signal transduction to adapt to phosphorus deficiency.

NIPK, a protein pseudokinase that interacts with the C subunit of the transcription factor NF-Y, is involved in rhizobial infection and nodule organogenesis.

Heterotrimeric Nuclear Factor Y (NF-Y) transcription factors are key regulators of the symbiotic program that controls rhizobial infection and nodule organogenesis. Using a yeast two-hybrid screening, we identified a putative protein kinase of Phaseolus vulgaris that interacts with the C subunit of the NF-Y complex. Physical interaction between NF-YC1 Interacting Protein Kinase (NIPK) and NF-YC1 occurs in the cytoplasm and the plasma membrane. Only one of the three canonical amino acids predicted to be required for catalytic activity is conserved in NIPK and its putative homologs from lycophytes to angiosperms, indicating that NIPK is an evolutionary conserved pseudokinase. Post-transcriptional silencing on NIPK affected infection and nodule organogenesis, suggesting NIPK is a positive regulator of the NF-Y transcriptional complex. In addition, NIPK is required for activation of cell cycle genes and early symbiotic genes in response to rhizobia, including NF-YA1 and NF-YC1. However, strain preference in co-inoculation experiments was not affected by NIPK silencing, suggesting that some functions of the NF-Y complex are independent of NIPK. Our work adds a new component associated with the NF-Y transcriptional regulators in the context of nitrogen-fixing symbiosis.

Transcriptomic analysis of Mesoamerican and Andean Phaseolus vulgaris accessions revealed mRNAs and lncRNAs associated with strain selectivity during symbiosis.

Legume plants establish a nitrogen-fixing symbiosis with soil bacteria known as rhizobia. Compatibility between legumes and rhizobia is determined at species-specific level, but variations in the outcome of the symbiotic process are also influenced by the capacity of the plant to discriminate and select specific strains that are better partners. We compared the transcriptional response of two genetically diverse accessions of Phaseolus vulgaris from Mesoamerica and South Andes to Rhizobium etli strains that exhibit variable degrees of symbiotic affinities. Our results indicate that the plant genotype is the major determinant of the transcriptional reprogramming occurring in roots at early stages of the symbiotic interaction. Differentially expressed genes (DEGs) regulated in the Mesoamerican and the Andean accessions in response to specific strains are different, but they belong to the same functional categories. The common and strain-specific transcriptional responses to rhizobia involve distinct transcription factors and cis-elements present in the promoters of DEGs in each accession, showing that diversification and domestication of common bean at different geographic regions influenced the evolution of symbiosis differently in each genetic pool. Quantitative PCR analysis validated our transcriptional datasets, which constitute a valuable source of coding and non-coding candidate genes to further unravel the molecular determinants governing the mechanisms by which plants select bacterial strains that produce a better symbiotic outcome.

Incorporation of 5-Bromo-2'-deoxyuridine into DNA and Proliferative Behavior of Cerebellar Neuroblasts: All That Glitters Is Not Gold.

The synthetic halogenated pyrimidine analog, 5-bromo-2'-deoxyuridine (BrdU), is a marker of DNA synthesis. This exogenous nucleoside has generated important insights into the cellular mechanisms of the central nervous system development in a variety of animals including insects, birds, and mammals. Despite this, the detrimental effects of the incorporation of BrdU into DNA on proliferation and viability of different types of cells has been frequently neglected. This review will summarize and present the effects of a pulse of BrdU, at doses ranging from 25 to 300 µg/g, or repeated injections. The latter, following the method of the progressively delayed labeling comprehensive procedure. The prenatal and perinatal development of the cerebellum are studied. These current data have implications for the interpretation of the results obtained by this marker as an index of the generation, migration, and settled pattern of neurons in the developing central nervous system. Caution should be exercised when interpreting the results obtained using BrdU. This is particularly important when high or repeated doses of this agent are injected. I hope that this review sheds light on the effects of this toxic maker. It may be used as a reference for toxicologists and neurobiologists given the broad use of 5-bromo-2'-deoxyuridine to label dividing cells.

Developmental timetables and gradients of neurogenesis in cerebellar Purkinje cells and deep glutamatergic neurons: A comparative study between the mouse and the rat.

The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([3 H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [3 H]TdR on embryonic days (E) 11-12, E12-13, E13-14, E14-15, E15-16, and E16-17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.

The PvNF-YA1 and PvNF-YB7 Subunits of the Heterotrimeric NF-Y Transcription Factor Influence Strain Preference in the Phaseolus vulgaris-Rhizobium etli Symbiosis.

Transcription factors of the Nuclear Factor Y (NF-Y) family play essential functions in plant development and plasticity, including the formation of lateral root organs such as lateral root and symbiotic nodules. NF-Ys mediate transcriptional responses by acting as heterotrimers composed of three subunits, NF-YA, NF-YB, and NF-YC, which in plants are encoded by relatively large gene families. We have previously shown that, in the Phaseolus vulgaris × Rhizobium etli interaction, the PvNF-YC1 subunit is involved not only in the formation of symbiotic nodules, but also in the preference exhibited by the plant for rhizobial strains that are more efficient and competitive in nodule formation. PvNF-YC1 forms a heterotrimer with the PvNF-YA1 and PvNF-YB7 subunits. Here, we used promoter:reporter fusions to show that both PvNF-YA1 and PvNF-YB7 are expressed in symbiotic nodules. In addition, we report that knock-down of PvNF-YA1 and its close paralog PvNF-YA9 abolished nodule formation by either high or low efficient strains and arrested rhizobial infection. On the other hand, knock-down of PvNF-YB7 only affected the symbiotic outcome of the high efficient interaction, suggesting that other symbiotic NF-YB subunits might be involved in the more general mechanisms of nodule formation. More important, we present functional evidence supporting that both PvNF-YA1 and PvNF-YB7 are part of the mechanisms that allow P. vulgaris plants to discriminate and select those bacterial strains that perform better in nodule formation, most likely by acting in the same heterotrimeric complex that PvNF-YC1.

Compatibility between Legumes and Rhizobia for the Establishment of a Successful Nitrogen-Fixing Symbiosis.

The root nodule symbiosis established between legumes and rhizobia is an exquisite biological interaction responsible for fixing a significant amount of nitrogen in terrestrial ecosystems. The success of this interaction depends on the recognition of the right partner by the plant within the richest microbial ecosystems on Earth, the soil. Recent metagenomic studies of the soil biome have revealed its complexity, which includes microorganisms that affect plant fitness and growth in a beneficial, harmful, or neutral manner. In this complex scenario, understanding the molecular mechanisms by which legumes recognize and discriminate rhizobia from pathogens, but also between distinct rhizobia species and strains that differ in their symbiotic performance, is a considerable challenge. In this work, we will review how plants are able to recognize and select symbiotic partners from a vast diversity of surrounding bacteria. We will also analyze recent advances that contribute to understand changes in plant gene expression associated with the outcome of the symbiotic interaction. These aspects of nitrogen-fixing symbiosis should contribute to translate the knowledge generated in basic laboratory research into biotechnological advances to improve the efficiency of the nitrogen-fixing symbiosis in agronomic systems.

Annotation, phylogeny and expression analysis of the nuclear factor Y gene families in common bean (Phaseolus vulgaris).

In the past decade, plant nuclear factor Y (NF-Y) genes have gained major interest due to their roles in many biological processes in plant development or adaptation to environmental conditions, particularly in the root nodule symbiosis established between legume plants and nitrogen fixing bacteria. NF-Ys are heterotrimeric transcriptional complexes composed of three subunits, NF-YA, NF-YB, and NF-YC, which bind with high affinity and specificity to the CCAAT box, a cis element present in many eukaryotic promoters. In plants, NF-Y subunits consist of gene families with about 10 members each. In this study, we have identified and characterized the NF-Y gene families of common bean (Phaseolus vulgaris), a grain legume of worldwide economical importance and the main source of dietary protein of developing countries. Expression analysis showed that some members of each family are up-regulated at early or late stages of the nitrogen fixing symbiotic interaction with its partner Rhizobium etli. We also showed that some genes are differentially accumulated in response to inoculation with high or less efficient R. etli strains, constituting excellent candidates to participate in the strain-specific response during symbiosis. Genes of the NF-YA family exhibit a highly structured intron-exon organization. Moreover, this family is characterized by the presence of upstream ORFs when introns in the 5' UTR are retained and miRNA target sites in their 3' UTR, suggesting that these genes might be subjected to a complex post-transcriptional regulation. Multiple protein alignments indicated the presence of highly conserved domains in each of the NF-Y families, presumably involved in subunit interactions and DNA binding. The analysis presented here constitutes a starting point to understand the regulation and biological function of individual members of the NF-Y families in different developmental processes in this grain legume.

Transcriptional regulators of legume-rhizobia symbiosis: nuclear factors Ys and GRAS are two for tango.

Transcription factors are DNA binding proteins that regulate gene expression. The nitrogen fixing symbiosis established between legume plants and soil bacteria is a complex interaction, in which plants need to integrate signals derived from the symbiont and the surrounding environment to initiate the developmental program of nodule organogenesis and the infection process. Several transcription factors that play critical roles in these processes have been reported in the past decade, including proteins of the GRAS and NF-Y families. Recently, we reported the characterization of a new GRAS domain containing-protein that interacts with a member of the C subunit of the NF-Y family, which plays an important role in nodule development and the progression of bacterial infection during the symbiotic interaction. The connection between transcription factors of these families highlights the significance of multimeric complexes in the fabulous capacity of plants to integrate and respond to multiple environmental stimuli.