Nitrogen Limitation Adaptation Research Articles

Differential Responses of Medicago truncatula NLA Homologs to Nutrient Deficiency and Arbuscular Mycorrhizal Symbiosis.

NITROGEN LIMITATION ADAPTATION (NLA), a plasma-membrane-associated ubiquitin E3 ligase, plays a negative role in the control of the phosphate transporter family 1 (PHT1) members in Arabidopsis and rice. There are three NLA homologs in the Medicago truncatula genome, but it has been unclear whether the function of these homologs is conserved in legumes. Here we investigated the subcellular localization and the responses of MtNLAs to external phosphate and nitrate status. Similar to AtNLA1, MtNLA1/MtNLA2 was localized in the plasma membrane and nucleus. MtNLA3 has three alternative splicing variants, and intriguingly, MtNLA3.1, the dominant variant, was not able to target the plasma membrane, whereas MtNLA3.2 and MtNLA3.3 were capable of associating with the plasma membrane. In contrast with AtNLA1, we found that MtNLAs were not affected or even upregulated by low-phosphate treatment. We also found that MtNLA3 was upregulated by arbuscular mycorrhizal (AM) symbiosis, and overexpressing MtNLA3.1 in Medicago roots resulted in a decrease in the transcription levels of STR, an essential gene for arbuscule development. Taken together, our results highlight the difference between MtNLA homologs and AtNLA1. Further characterization will be required to reveal the regulation of these genes and their roles in the responses to external nutrient status and AM symbiosis.

Diverse functions of GmNLA1 members in controlling phosphorus homeostasis highlight coordinate response of soybean to nitrogen and phosphorus availability

Nitrogen (N) and phosphorus (P) are two essential mineral nutrients for plant growth, which are required in relative high amount in plants. Plants have evolved a series of strategies for coordinately acquiring and utilizing N and P. However, physiological and molecular mechanisms underlying of N and P interactions remain largely unclear in soybean (Glycine max). In this study, interactions of N and P were demonstrated in soybean as reflected by significant increases of phosphate (Pi) concentration in both leaves and roots by N deficiency under Pi sufficient conditions. A total of four nitrogen limitation adaptation (NLA), encoding RING-type E3 ubiquitin ligase were subsequently identified in soybean genome. Among them, transcription of GmNLA1-1 and GmNLA1-3 was decreased in soybean by N starvation under Pi sufficient conditions, not for GmNLA1-2. Suppression of all three GmNLA1 members was able to increase Pi concentration regardless of the P and N availability in the growth medium, but decrease fresh weight under normal conditions in soybean hairy roots. However, comparted to changes in control lines at two N levels, N deficiency only resulted in a relatively higher increase of Pi concentration in GmNLA1-1 or GmNLA1-3 suppression lines, strongly indicating that GmNLA1-1 and GmNLA1-3 might regulate P homeostasis in soybean response to N starvation. Taken together, our result suggest that redundant and diverse functions present in GmNLA1 members for soybean coordinate responses to P and N availability, which mediate P homeostasis.

Inositol polyphosphates-regulated polyubiquitination of PHR1 by NLA E3 ligase during phosphate starvation response in Arabidopsis.

Phosphate (Pi) availability is a major factor limiting plant growth and development. The key transcription factor controlling Pi-starvation response (PSR) is PHOSPHATE STARVATION RESPONSE 1 (PHR1) whose transcript levels do not change with changes in Pi levels. However, how PHR1 stability is regulated at the post-translational level is relatively unexplored in Arabidopsis thaliana. Inositol polyphosphates (InsPn) are important signal molecules that promote the association of stand-alone SPX domain proteins with PHR1 to regulate PSR. Here, we show that NITROGEN LIMITATION ADAPTATION (NLA) E3 ligase can associate with PHR1 through its conserved SPX domain and polyubiquitinate PHR1 invitro. The association with PHR1 and its ubiquitination is enhanced by InsP6 but not by InsP5. Analysis of InsPn-related mutants and an overexpression plant shows PHR1 levels are more stable in itpk4-1 and vih2-4/VIH1amiRNA but less stable in ITPK4 overexpression plants. Under Pi-deficient conditions, nla seedlings contain high PHR1 levels, display long root hair and accumulate anthocyanin in shoots phenocopying PHR1 overexpression plants. By contrast, NLA overexpression plants phenocopy phr1 whose phenotypes are opposite to those of nla. Our results suggest NLA functions as a negative regulator of Pi response by modulating PHR1 stability and the NLA/PHR1 association depends on InsPn levels.

Genome-Wide Identification and Functional Characterization Reveals the Pivotal Roles of BnaA8.ATG8F in Salt Stress Tolerance and Nitrogen Limitation Adaptation in Allotetraploid Rapeseed.

Autophagy is a common physiological process in organisms, including higher plants. The ATG8 subfamily, the core member of the autophagy-related gene (ATG) family, plays a key role in plant growth and development and nutrient stress responses. However, the core ATG8 homologs and their roles in stress resistance remain elusive in allotetraploid rapeseed (AACC, Brassica napus L.). In this study, we identified 29 ATG8 subgroup members, consisting of three phylogenetic clades, based on the analysis of genomic annotation and conserved motifs. Differential transcriptional responses of BnaATG8s to salt stress, nitrogen limitation, and other nutrient stresses were investigated, and we identified BnaA8.ATG8F as the core ATG8 member through gene co-expression network analysis. Decreased BnaA8.ATG8F expression repressed the salt tolerance of transgenic rapeseed plants by significantly reducing the root Na+ retention under salt stress. Moreover, downregulation of BnaA8.ATG8F increased nitrogen (N) limitation sensitivity of transgenic rapeseed plants through decreasing N uptake, translocation, and enhancing N remobilization under nitrogen starvation. In summary, we identified the core ATG8 homologs and characterized their physiological and molecular mechanisms underlying salt stress tolerance and nitrogen limitation adaptation. Our results may provide elite genetic resources for the genetic improvement of nutrient stress tolerance in rapeseed.

Loss-of-function of NITROGEN LIMITATION ADAPTATION confers disease resistance in Arabidopsis by modulating hormone signaling and camalexin content

Phosphorus is an important macronutrient required for plant growth and development. It is absorbed by the roots in the form of inorganic phosphate (Pi). Under Pi limiting conditions, plants activate the Phosphate Starvation Response (PSR) system to enhance Pi acquisition. The NITROGEN LIMITATION ADAPTION (NLA) gene is a component of the Arabidopsis PSR, and its expression is post-transcriptionally regulated by miR827. We show that loss-of-function of NLA and MIR827 overexpression increases Pi level and enhances resistance to infection by the fungal pathogen Plectosphaerella cucumerina in Arabidopsis. Upon pathogen infection, high Pi plants (e.g. nla plants and wild type plants grown under high Pi supply) showed enhanced callose deposition. High Pi plants also exhibited superinduction of camalexin biosynthesis genes which is consistent with increased levels of camalexin during pathogen infection. Pathogen infection and treatment with fungal elicitors, triggered up-regulation of MIR827 and down-regulation of NLA expression. Under non-infection conditions, the nla plants showed increased levels of SA and JA compared with wild type plants, their levels further increasing upon pathogen infection. Overall, the outcomes of this study suggest that NLA plays a role in Arabidopsis immunity, while supporting convergence between Pi signaling and immune signaling in Arabidopsis.

Review of NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity

Review of NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity

Genome-Wide Differential DNA Methylation and miRNA Expression Profiling Reveals Epigenetic Regulatory Mechanisms Underlying Nitrogen-Limitation-Triggered Adaptation and Use Efficiency Enhancement in Allotetraploid Rapeseed.

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

Integrated Transcriptional and Proteomic Profiling Reveals Potential Amino Acid Transporters Targeted by Nitrogen Limitation Adaptation.

Nitrogen (N) is essential for plant growth and crop productivity. Organic N is a major form of remobilized N in plants’ response to N limitation. It is necessary to understand the regulatory role of N limitation adaption (NLA) in organic N remobilization for this adaptive response. Transcriptional and proteomic analyses were integrated to investigate differential responses of wild-type (WT) and nla mutant plants to N limitation and to identify the core organic N transporters targeted by NLA. Under N limitation, the nla mutant presented an early senescence with faster chlorophyll loss and less anthocyanin accumulation than the WT, and more N was transported out of the aging leaves in the form of amino acids. High-throughput transcriptomic and proteomic analyses revealed that N limitation repressed genes involved in photosynthesis and protein synthesis, and promoted proteolysis; these changes were higher in the nla mutant than in the WT. Both transcriptional and proteomic profiling demonstrated that LHT1, responsible for amino acid remobilization, were only significantly upregulated in the nla mutant under N limitation. These findings indicate that NLA might target LHT1 and regulate organic N remobilization, thereby improving our understanding of the regulatory role of NLA on N remobilization under N limitation.

Differential expression of miRNAs and their targets in wax-deficient rapeseed

The cuticle of a plant, composed of cutin and wax, is the outermost hydrophobic layer covering the epidermis of all its aerial organs, protecting it from many abiotic and biotic stresses. The biosynthesis and regulation pathways of wax components have been well studied, whereas there are fewer reports on the small RNA-involved post-transcriptional regulation of wax biosynthesis in plants, particularly in Brassica napus. Previously, we conducted a study on a glossy mutant of rapeseed, and we assumed that there was a dominant repressor to inhibit the expression of wax-related genes. To verify this hypothesis and investigate the function of small RNAs in wax biosynthesis in B. napus, we constructed four small RNA libraries from the stem epidermis of wax-deficient mutant and wild-type plants for sequencing. Subsequently, 43,840,451 clean reads were generated and 24 nt sequences represented the dominant percentage. In total, 300 unique known miRNAs were identified and eight of them showed differential expression. In addition, the expression levels of six novel miRNAs were altered. Surprisingly, we found that four up-regulated miRNAs in the wax-deficient plants, bna-miR408b-5p, bna-miR165b-5p, bna-miR160a-3p, and bna-miR398-5p, were all complementary strands of their corresponding mature strands. Stem-loop qRT-PCR verified that the expression of bna-miR165a-5p was increased in the mutant stems, while its putative target, BnaA06g40560D (CYP96A2), was down-regulated. In addition, the expression of bna-miR827a was detected to be down-regulated in glossy mutant. 5′ RACE experimental data showed that bna-miR827a cleaves three NITROGEN LIMITATION ADAPTATION (NLA) genes (BnaC08g45940D, BnaA10g01450D and BnaC05g01480D). The down-regulation of bna-miR827a resulted in decreased cleavage on its targets, and led to the up-regulation of its targets, especially BnaA10g01450D gene. These results showed that bna-miR165a-5p might participate in wax biosynthesis process by regulating its putative target BnaA06g40560D (CYP96A2). The expression levels of a phosphate (Pi)-related miRNA, bna-miR827a, and its target genes were affected in wax-deficient rapeseeds. These results will promote the study of post-transcriptional regulation mechanisms of wax biosynthesis in B. napus and provide new directions for further research.

Arabidopsis ubiquitin-specific proteases UBP12 and UBP13 shape ORE1 levels during leaf senescence induced by nitrogen deficiency.

Nitrogen deficiency (-N) in plants triggers leaf senescence which is regulated by the transcription factor ORE1. Little is known about post-translational regulation of ORE1 in this process. Here, we show that UBP12/UBP13 (ubiquitin-specific protease 12/13) antagonize the action of NLA (nitrogen limitation adaptation) E3 ligase to maintain ORE1 homeostasis. In vitro pull-down and in vivo co-immunoprecipitation assays demonstrated specific binding between UBP12/UBP13 and ORE1. We further analyzed in various genotypes total Chl content and expression levels of senescence-related genes under -N conditions. We found that UBP12/UBP13 can deubiquitinate polyubiquitinated ORE1 in vitro and increase the stability of ORE1 in vivo in MG132/cycloheximide-chase experiments. Plants overexpressing UBP12/UBP13 display accelerated leaf senescence which is reversed by the ore1 mutation. By contrast, the senescence phenotype of plants overexpressing ORE1 is exacerbated by UBP12/UBP13 overexpression. The expression of senescence-related genes tracks the senescence phenotype. ORE1 protein levels can be elevated by UBP12/UBP13 overexpression but decreased in ubp12-2w/13-3. In conclusion, UBP12/UBP13 deubiquitinate ORE1 to stabilize this transcription factor and promote its activity as a positive regulator for leaf senescence under -N conditions. Our study shows that UBP12/UBP13 counteracts the effect of NLA E3 ligase to accelerate leaf senescence under nitrogen starvation.

Arabidopsis NITROGEN LIMITATION ADAPTATION regulates ORE1 homeostasis during senescence induced by nitrogen deficiency.

Nitrogen is an important macronutrient in plants and its deficiency induces rapid leaf senescence. Two genes, ORE1 and NITROGEN LIMITATION ADAPTATION (NLA), have been implicated in regulating the senescence process but their relationship is unclear1,2. Here, we show that nla and pho2 (also known as ubc24) plants develop rapid leaf senescence under nitrogen-starvation condition, whereas ore1 and nla/ore1 and pho2 (ubc24)/ore1 plants stay green. These results suggest that ORE1 acts downstream of NLA and PHO2 (UBC24). NLA interacts with ORE1 in the nucleus and regulates its stability through polyubiquitination using PHO2 (UBC24) as the E2 conjugase. Our findings identified ORE1 as a downstream target of NLA/PHO2 (UBC24) and showed that post-translational regulation of ORE1 levels determines leaf senescence during nitrogen deficiency.

Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two different subcellular sites (the plasma membrane and the nucleus), and contains four distinct domains: (i) a RING domain that mediates degradation of phosphate transporters at the plasma membrane; (ii) an SPX domain that facilitates NLA’s interaction with the phosphate transporters, and also exists in other proteins that regulate the nuclear transcription factors that control the phosphate starvation response pathway; (iii) a linker domain that lies between the RING and SPX domains; and (iv) a C-terminal domain, which, like the linker region, is of unknown function. Here we carried out a mutational analysis of NLA, which indicated that all the domains are not only essential for proper functioning of the protein, but also mediate its localization to the plasma membrane and (or) nucleus, as well as to different subdomains within the nucleus. Overall, the results provide new insights to the distinct protein motifs within NLA and the role(s) that this protein serves at different subcellular sites with respect to the regulation of nitrogen-dependent phosphate homeostasis as well as other possible physiological functions.

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.

OsNLA1, a RING-type ubiquitin ligase, maintains phosphate homeostasis in Oryza sativa via degradation of phosphate transporters.

Inorganic phosphate (Pi) transporters (PTs) play vital roles in Pi uptake and translocation in plants. Under Pi sufficient conditions, PTs are degraded to prevent excess Pi accumulation. The mechanisms targeting PTs for degradation are not fully elucidated. In this study, we found that the Oryza sativa (rice) ortholog of Arabidopsis thaliana nitrogen limitation adaptation (NLA), OsNLA1 protein, a RING-type E3 ubiquitin-ligase, was predominantly localized in the plasma membrane, and could interact with rice phosphate transporters OsPT2 and OsPT8. Mutation of the 265th cysteine residue in OsNLA1 that was required for ubiquitination prevented breakdown of OsPT2/PT8, suggesting OsNLA1 targeted OsPT2/PT8 for degradation. Mutation in OsNLA1 (osnla1) led to a significant increase of Pi concentration in leaves in a nitrate-independent manner. Overexpression of OsNLA1 or repression of OsPT2/PT8 restored the high leaf Pi concentration in osnla1 mutants to a level similar to that of wild-type plants. In contrast to what has been observed in Arabidopsis, the transcript abundance of OsNLA1 did not decrease under Pi limited conditions or in OsmiR827 (microRNA827)- or OsPHR2 (PHOSPHATE STARVATION RESPONSE 2)-overexpressing transgenic lines. Moreover, there was no interaction of OsNLA1 and OsPHO2, an E2 ubiquitin-conjugase, suggesting that OsPHO2 was not the partner of OsNLA1 involved in ubiquitin-mediated PT degradation. Our results show that OsNLA1 is involved in maintaining phosphate homeostasis in rice by mediating the degradation of OsPT2 and OsPT8, and OsNLA1 differs from the ortholog in Arabidopsis in several aspects.

Nitrogen Limitation Adaptation (NLA) is involved in source-to-sink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis.

Recent studies on nitrate transporters (NRTs) have greatly increased our knowledge of the mechanisms regulating nitrogen (N) homeostasis in plants. However, an understanding of how these NRTs are regulated is still lacking. The nitrogen limitation adaptation (nla) mutant is hypersensitive to N limitation. In the nla mutant, 15 N-nitrate spotted on old leaves preferentially accumulated in the youngest leaves. Analysis of leaf cross-sections indicated that NLA expression was expressed in the sieve element and companion cell system. The results of bimolecular fluorescence complementation (BiFC), split-ubiquitin yeast two-hybrid and co-immunoprecipitation (CoIP) assays demonstrated that NLA interacts with NRT1.7 in the plasma membrane. The following findings suggest that NLA directs the ubiquitination of NRT1.7: the down-regulation of NRT1.7 protein abundance in 35S::NLA/35S::Myc-NRT1.7 double transgenic plants compared with 35S::Myc-NRT1.7 transgenic plants; the up-regulation of NRT1.7 protein abundance in the nla mutant compared with wild-type plants; and the direct degradation of truncated NRT1.7 recombinant protein by NLA. Furthermore, analysis of NLA and NRT1.7 protein abundance in mirna827 knock-out plants showed that N deficiency-guided translational repression of NLA depends on miRNA827. Our findings reveal that plants regulate source-to-sink remobilization of nitrate by the ubiquitin-mediated post-translational regulatory pathway.

Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility.

MicroRNAs (miRNAs) are a major class of small non-coding RNAs with emerging functions in biotic and abiotic interactions. Here, we report on a new functional role of Arabidopsis miR827 and its NITROGEN LIMITATION ADAPTATION (NLA) target gene in mediating plant susceptibility to the beet cyst nematode Heterodera schachtii. Cyst nematodes are sedentary endoparasites that induce the formation of multinucleated feeding structures termed syncytia in the roots of host plants. Using promoter:GUS fusion assays we established that miR827 was activated in the initial feeding cells and this activation was maintained in the syncytium during all sedentary stages of nematode development. Meanwhile, the NLA target gene, which encodes an ubiquitin E3 ligase enzyme, was post-transcriptionally silenced in the syncytium to permanently suppress its activity during all nematode parasitic stages. Overexpression of miR827 in Arabidopsis resulted in hyper-susceptibility to H.schachtii. In contrast, inactivation of miR827 activity through target mimicry or by overexpression a miR827-resistant cDNA of NLA produced the opposite phenotype of reduced plant susceptibility to H.schachtii. Gene expression analysis of several pathogenesis-related genes together with Agrobacterium-mediated transient expression in Nicotiana benthamiana provided strong evidence that miR827-mediated downregulation of NLA to suppress basal defense pathways. In addition, using yeast two-hybrid screens we identified several candidates of NLA-interacting proteins that are involved in a wide range of biological processes and molecular functions, including three pathogenesis-related proteins. Taken together, we conclude that nematode-activated miR827 in the syncytium is necessary to suppress immune responses in order to establish infection and cause disease.

NITROGEN LIMITATION ADAPTATION Recruits PHOSPHATE2 to Target the Phosphate Transporter PT2 for Degradation during the Regulation of Arabidopsis Phosphate Homeostasis

The NITROGEN LIMITATION ADAPTION (NLA) gene was initially shown to function in nitrogen limitation responses; however, recent work shows that the nla mutant hyperaccumulates Pi, phenocopying the Pi signaling mutant pho2. PHO2 encodes a putative E2 conjugase, UBC24. Here, we show that NLA is an E3 ligase that specifically requires UBC24 for polyubiquitination in Arabidopsis thaliana. Among five members of the Pht1 Pi-transporter family tested, NLA associates only with PT2 (Pht1;4). The NLA-UBC24 pair mediates polyubiquitination of PT2 but not PT1. Posttranslational decay of PT2 at high Pi is blocked in pho2 and inhibited by MG132, indicating the requirement of UBC24 and 26S proteasomes. Consistent with NLA/UBC24 function, induced NLA expression causes a UBC24-dependent decrease in PT2 levels. Confocal microscopy of fusion proteins revealed an NLA/PT2 interaction at the plasma membrane. Collectively, these results show that under Pi-replete conditions, NLA and UBC24 target the PT2 transporter for destruction. During the Pi deprivation response, NLA and PHO2 transcripts are cleaved by miR399 and miR827, respectively, and our results suggest that this downregulation relieves the posttranslational repression of PT2, allowing it to accumulate and participate in Pi uptake. Our work provides additional molecular details describing Pi signaling/homeostasis regulation by identifying NLA and UBC24 as partners and PT2 as one of their downstream targets.

Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis.

Members of the Arabidopsis thaliana phosphate transporter1 (PHT1) family are key players in acquisition of Pi from the rhizosphere, and their regulation is indispensable for the maintenance of cellular Pi homeostasis. Here, we reveal posttranslational regulation of Pi transport through modulation of degradation of PHT1 proteins by the RING-type ubiquitin E3 ligase, nitrogen limitation adaptation (NLA). Loss of function of NLA caused high Pi accumulation resulting from increases in the levels of several PHT1s at the protein rather than the transcript level. Evidence of decreased endocytosis and ubiquitination of PHT1s in nla mutants and interaction between NLA and PHT1s in the plasma membranes suggests that NLA directs the ubiquitination of plasma membrane-localized PHT1s, which triggers clathrin-dependent endocytosis followed by endosomal sorting to vacuoles. Furthermore, different subcellular localization of NLA and phosphate2 (pho2; a ubiquitin E2 conjugase) and the synergistic effect of the accumulation of PHT1s and Pi in nla pho2 mutants suggest that they function independently but cooperatively to regulate PHT1 protein amounts. Intriguingly, NLA and PHO2 are the targets of two Pi starvation-induced microRNAs, miR827 and miR399, respectively. Therefore, our findings uncover modulation of Pi transport activity in response to Pi availability through the integration of a microRNA-mediated posttranscriptional pathway and a ubiquitin-mediated posttranslational regulatory pathway.

Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in arabidopsis.

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion.

Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene.

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.