Bikram Datt Pant

PHO2, MicroRNA399, and PHR1 Define a Phosphate-Signaling Pathway in Plants

Inorganic phosphate (Pi)-signaling pathways in plants are still largely unknown. The Arabidopsis (Arabidopsis thaliana) pho2 mutant overaccumulates Pi in leaves in Pi-replete conditions. Micrografting revealed that a pho2 root genotype is sufficient to yield leaf Pi accumulation. In pho2 mutants, Pi does not repress a set of Pi starvation-induced genes, including AtIPS1, AT4, and Pi transporters Pht1;8 and Pht1;9. Map-based cloning identified PHO2 as At2g33770, an unusual E2 conjugase gene. It was recently shown that Pi deprivation induces mature microRNA (miRNA [miR399]) and that overexpression of miR399 in Pi-replete conditions represses E2 conjugase expression and leads to high leaf Pi concentrations, thus phenocopying pho2. We show here that miR399 primary transcripts are also strongly induced by low Pi and rapidly repressed after addition of Pi. PHO2 transcripts change reciprocally to miR399 transcripts in Pi-deprived plants and in miR399 overexpressers. However, responses after Pi readdition and in β-glucuronidase reporter lines suggest that PHO2 expression is also regulated by Pi in a manner unrelated to miR399-mediated transcript cleavage. Expression of miR399 was strongly reduced in Pi-deprived Arabidopsis phr1 mutants, and a subset of Pi-responsive genes repressed in Pi-deprived phr1 mutants was up-regulated in Pi-replete pho2 mutants. This places miR399 and PHO2 in a branch of the Pi-signaling network downstream of PHR1. Finally, putative PHO2 orthologs containing five miR399-binding sites in their 5′-untranslated regions were identified in other higher plants, and Pi-dependent miR399 expression was demonstrated in rice (Oryza sativa), suggesting a conserved regulatory mechanism.

MicroRNA399 is a long‐distance signal for the regulation of plant phosphate homeostasis

The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively over-expressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA.

Identification of Nutrient-Responsive Arabidopsis and Rapeseed MicroRNAs by Comprehensive Real-Time Polymerase Chain Reaction Profiling and Small RNA Sequencing

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Arabidopsis miR156 Regulates Tolerance to Recurring Environmental Stress through SPL Transcription Factors

The authors show that a well-conserved miRNA-transcription factor module implicated previously in developmental control regulates responses to repeated heat stress. They provide a conceptual framework for the integration of environmental stress responses with development to optimize growth under natural conditions. Plants are sessile organisms that gauge stressful conditions to ensure survival and reproductive success. While plants in nature often encounter chronic or recurring stressful conditions, the strategies to cope with those are poorly understood. Here, we demonstrate the involvement of ARGONAUTE1 and the microRNA pathway in the adaptation to recurring heat stress (HS memory) at the physiological and molecular level. We show that miR156 isoforms are highly induced after HS and are functionally important for HS memory. miR156 promotes sustained expression of HS-responsive genes and is critical only after HS, demonstrating that the effects of modulating miR156 on HS memory do not reflect preexisting developmental alterations. miR156 targets SPL transcription factor genes that are master regulators of developmental transitions. SPL genes are posttranscriptionally downregulated by miR156 after HS, and this is critical for HS memory. Altogether, the miR156-SPL module mediates the response to recurring HS in Arabidopsis thaliana and thus may serve to integrate stress responses with development.

Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability

• Reduced oxygen availability is not only associated with flooding, but occurs also during growth and development. It is largely unknown how hypoxia is perceived and what signaling cascade is involved in activating adaptive responses. • We analysed the expression of over 1900 transcription factors (TFs) and 180 microRNA primary transcripts (pri-miRNAs) in Arabidopsis roots exposed to different hypoxic conditions by means of quantitative PCR. We also analysed the promoters of genes induced by hypoxia with respect to over-represented DNA elements that can act as potential TF binding sites and their in vivo interaction was verified. • We identified various subsets of TFs that responded differentially through time and in an oxygen concentration-dependent manner. The regulatory potential of selected TFs and their predicted DNA binding elements was validated. Although the expression of pri-miRNAs was differentially regulated under hypoxia, only one corresponding mature miRNA changed accordingly. Putative target transcripts of the miRNAs were not significantly affected. • Our results show that the regulation of hypoxia-induced genes is controlled via simultaneous interaction of various combinations of TFs. Under anoxic conditions, an additional set of TFs is induced. Regulation of gene expression via miRNAs appears to play a minor role during hypoxia.

Expression pattern suggests a role of MiR399 in the regulation of the cellular response to local Pi increase during arbuscular mycorrhizal symbiosis.

Many plants improve their phosphate (Pi) availability by forming mutualistic associations with arbuscular mycorrhizal (AM) fungi. Pi-repleted plants are much less colonized by AM fungi than Pi-depleted plants. This indicates a link between plant Pi signaling and AM development. MicroRNAs (miR) of the 399 family are systemic Pi-starvation signals important for maintenance of Pi homeostasis in Arabidopsis thaliana and might also qualify as signals regulating AM development in response to Pi availability. MiR399 could either represent the systemic low-Pi signal promoting or required for AM formation or they could act as counter players of systemic Pi-availability signals that suppress AM symbiosis. To test either of these assumptions, we analyzed the miR399 family in the AM-capable plant model Medicago truncatula and could experimentally confirm 10 novel MIR399 genes in this species. Pi-depleted plants showed increased expression of mature miR399 and multiple pri-miR399, and unexpectedly, levels of five of the 15 pri-miR399 species were higher in leaves of mycorrhizal plants than in leaves of nonmycorrhizal plants. Compared with nonmycorrhizal Pi-depleted roots, mycorrhizal roots of Pi-depleted M. truncatula and tobacco plants had increased Pi contents due to symbiotic Pi uptake but displayed higher mature miR399 levels. Expression levels of MtPho2 remained low and PHO2-dependent Pi-stress marker transcript levels remained high in these mycorrhizal roots. Hence, an AM symbiosis-related signal appears to increase miR399 expression and decrease PHO2 activity. MiR399 overexpression in tobacco suggested that miR399 alone is not sufficient to improve mycorrhizal colonization supporting the assumption that, in mycorrhizal roots, increased miR399 are necessary to keep the MtPho2 expression and activity low, which would otherwise increase in response to symbiotic Pi uptake.

Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3′ and 5′ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5′ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

The transcription factor PHR1 regulates lipid remodeling and triacylglycerol accumulation in Arabidopsis thaliana during phosphorus starvation

Highlight This study reveals that the transcription factor PHR1 controls phospholipid/glycolipid substitution during P starvation, and that Arabidopsis accumulates triacylglycerol during P starvation. In roots this phenotype is also under control of PHR1.

Expression of sucrose transporter cDNAs specifically in companion cells enhances phloem loading and long-distance transport of sucrose, but leads to an inhibition of growth and the perception of a phosphate limitation

Increased expression of selected sucrose/proton symporters above wild-type levels in phloem companion cells enhanced phloem loading and long-distant transport but unexpectedly resulted in an inhibition of growth that is associated with perception of a phosphate limitation. Sucrose (Suc) is the predominant form of carbon transported through the phloem from source to sink organs and is also a prominent sugar for short-distance transport. In all streptophytes analyzed, Suc transporter genes (SUTs or SUCs) form small families, with different subgroups evolving distinct functions. To gain insight into their capacity for moving Suc in planta, representative members of each clade were first expressed specifically in companion cells of Arabidopsis (Arabidopsis thaliana) and tested for their ability to rescue the phloem-loading defect caused by the Suc transporter mutation, Atsuc2-4. Sequence similarity was a poor indicator of ability: Several genes with high homology to AtSUC2, some of which have phloem-loading functions in other eudicot species, did not rescue the Atsuc2-4 mutation, whereas a more distantly related gene, ZmSUT1 from the monocot Zea mays, did restore phloem loading. Transporter complementary DNAs were also expressed in the companion cells of wild-type Arabidopsis, with the aim of increasing productivity by enhancing Suc transport to growing sink organs and reducing Suc-mediated feedback inhibition on photosynthesis. Although enhanced Suc loading and long-distance transport was achieved, growth was diminished. This growth inhibition was accompanied by increased expression of phosphate (P) starvation-induced genes and was reversed by providing a higher supply of external P. These experiments suggest that efforts to increase productivity by enhancing sugar transport may disrupt the carbon-to-P homeostasis. A model for how the plant perceives and responds to changes in the carbon-to-P balance is presented.

Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.