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The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout ofenod93in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed inenod93. However, a progressive loss of ADP-dependent respiration rate was observed inenod93mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher inenod93but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of wholeenod93seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots ofenod93compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes.Highlight significanceWe identify the enigmatic early nodulin ENOD93 gene family as the plant homolog of the N-terminal regulatory domain of the yeast RESPIRATORY SUPERCOMPLEX 2 (RCF2) of the mitochondrial oxidative phosphorylation system and provide biochemical and physiological evidence of its role in plant ATP production, broadly explaining the role of ENOD93 in plants.

Motivation Three-way data structures, characterized by three entities, the units, the variables and the occasions, are frequent in biological studies. In RNA sequencing, three-way data structures are obtained when high-throughput transcriptome sequencing data are collected for n genes across p conditions at r occasions. Matrix variate distributions offer a natural way to model three-way data and mixtures of matrix variate distributions can be used to cluster three-way data. Clustering of gene expression data is carried out as means of discovering gene co-expression networks. Results In this work, a mixture of matrix variate Poisson-log normal distributions is proposed for clustering read counts from RNA sequencing. By considering the matrix variate structure, full information on the conditions and occasions of the RNA sequencing dataset is simultaneously considered, and the number of covariance parameters to be estimated is reduced. We propose three different frameworks for parameter estimation: a Markov chain Monte Carlo-based approach, a variational Gaussian approximation-based approach, and a hybrid approach. Various information criteria are used for model selection. The models are applied to both real and simulated data, and we demonstrate that the proposed approaches can recover the underlying cluster structure in both cases. In simulation studies where the true model parameters are known, our proposed approach shows good parameter recovery. Availability and implementation The GitHub R package for this work is available at https://github.com/anjalisilva/mixMVPLN and is released under the open source MIT license.

This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the 'bibenzyls'. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this 'bibenzyl synthase' enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing 'parts prospecting' inventory that supports the rational metabolic engineering of natural product therapeutics.

In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for an O-methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ► chrysoeriol ► cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

Studies have indicated that the carbon starvation response leads to the reprogramming of the transcriptome and metabolome, and many genes, including several important regulators, such as the group S1 basic leucine zipper transcription factors (TFs) bZIP1, bZIP11 and bZIP53, the SNAC-A TF ATAF1, etc., are involved in these physiological processes. Here, we show that the SNAC-A TF ANAC032 also plays important roles in this process. The overexpression of ANAC032 inhibits photosynthesis and induces reactive oxygen species accumulation in chloroplasts, thereby reducing sugar accumulation and resulting in carbon starvation. ANAC032 reprograms carbon and nitrogen metabolism by increasing sugar and amino acid catabolism in plants. The ChIP-qPCR and transient dual-luciferase reporter assays indicated that ANAC032 regulates trehalose metabolism via the direct regulation of TRE1 expression. Taken together, these results show that ANAC032 is an important regulator of the carbon/energy status that represses photosynthesis to induce carbon starvation.

Nitrate promotes seed germination at low concentrations in many plant species, and functions as both a nutrient and a signal. As a nutrient, it is assimilated via nitrite to ammonium, which is then incorporated into amino acids. Nitrate reductase (NR) catalyses the reduction of nitrate to nitrite, the committed step in the assimilation. Seed sensitivity to nitrate is affected by other environmental factors, such as light and after-ripening, and by genotypes. Mode of nitrate action in seed germination has been well documented in Arabidopsis thaliana and the hedge mustard Sisymbrium officinale. In these species nitrate promotes seed germination independent of its assimilation by NR, suggesting that it acts as a signal to stimulate germination. In Arabidopsis, maternally applied nitrate affects the degree of primary dormancy in both wild-type and mutants defective in NR. This indicates that nitrate acts not only during germination, but also during seed development to negatively regulate primary dormancy. Functional genomics studies in Arabidopsis have revealed that nitrate elicits downstream events similar to other germination stimulators, such as after-ripening, light and stratification, suggesting that these distinct environmental signals share the same target(s). In Arabidopsis, the NIN-like protein 8 (NLP8) transcription factor, which acts downstream of nitrate signalling, induces nitrate-dependent gene expression. In particular, a gene encoding the abscisic acid (ABA) catabolic enzyme CYP707A2 is directly regulated by NLP8. This regulation triggers a nitrate-induced ABA decrease that permits seed germination. This review article summarizes an update of our current understanding of the regulation of seed dormancy and germination by nitrate.

Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem-loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.

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.

Agronomic traits controlling the formation, architecture and physiology of source and sink organs are main determinants of rice productivity. Semi-dwarf rice varieties with low tiller formation but high seed production per panicle and dark green and thick leaves with prolonged source activity are among the desirable traits to further increase the yield potential of rice. Here, we report the functional characterization of a zinc finger transcription factor, OsGATA12, whose overexpression causes increased leaf greenness, reduction of leaf and tiller number, and affects yield parameters. Reduced tillering allowed testing the transgenic plants under high density which resulted in significantly increased yield per area and higher harvest index compared to wild-type. We show that delayed senescence of transgenic plants and the corresponding longer stay-green phenotype is mainly due to increased chlorophyll and chloroplast number. Further, our work postulates that the increased greenness observed in the transgenic plants is due to more chlorophyll synthesis but most significantly to decreased chlorophyll degradation, which is supported by the reduced expression of genes involved in the chlorophyll degradation pathway. In particular we show evidence for the down-regulation of the STAY GREEN RICE gene and in vivo repression of its promoter by OsGATA12, which suggests a transcriptional repression function for a GATA transcription factor for prolonging the onset of senescence in cereals.

NAC (NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped cotyledon)) proteins are one of the largest families of plant-specific transcription factors, and this family is present in a wide range of land plants. Here, we have investigated the role of ANAC046 in the regulation of suberin biosynthesis and deposition in Arabidopsis. Subcellular localization and transcriptional activity assays showed that ANAC046 localizes in the nucleus, where it functions as a transcription activator. Analysis of the PANAC046:GUS lines revealed that ANAC046 is mainly expressed in the root endodermis and periderm, and is also induced in leaves by wounding. The transgenic lines overexpressing ANAC046 exhibited defective surfaces on the aerial plant parts compared to the wild-type (WT) as characterized by increased permeability for Toluidine blue stain and greater chlorophyll leaching. Quantitative RT-PCR analysis showed that the expression of suberin biosynthesis genes was significantly higher in the roots and leaves of overexpression lines compared to the WT. The biochemical analysis of leaf cuticular waxes showed that the overexpression lines accumulated 30% more waxes than the WT. Concurrently, overexpression lines also deposited almost twice the amount of suberin content in their roots compared with the WT. Taken together, these results showed that ANAC046 is an important transcription factor that promotes suberin biosynthesis in Arabidopsis thaliana roots.

Members of the NAC transcription factor family have been implicated in the regulation of different processes of plant development including senescence. In this study, the role of ANAC032 is analyzed in Arabidopsis thaliana (Col-0). ANAC032 is shown to act as a transcriptional activator and its expression is induced in senescing leaves as well as in dark-treated detached leaves. Analysis of transgenic overexpressors (OXs) and chimeric repressors (SRDXs) of ANAC032 indicates that ANAC032 positively regulates age-dependent and dark-induced leaf senescence. Quantitative real-time PCR analysis showed that ANAC032 regulates leaf senescence mainly through the modulation of expression of the senescence-associated genes AtNYE1, SAG113 and SAUR36/SAG201, which are involved in Chl degradation, and ABA and auxin promotion of senescence, respectively. In addition, ANAC032 expression is induced by a range of oxidative and abiotic stresses. As a result, ANAC032 overexpression lines exhibited enhanced leaf senescence when challenged with different oxidative (3-aminotriazole, fumonisin B1 and high light) and abiotic stress (osmotic and salinity) conditions compared with the wild type. In contrast, ANAC032 SRDX lines displayed the opposite phenotype. ANAC032 transgenic lines showed altered 2,4-D-mediated root tip swelling and root inhibition responses when compared with the wild type. The altered response to auxin, oxidative and abiotic stress treatments in ANAC032 transgenic lines involves differential accumulation of H2O2 compared with the wild type. Taken together, these results indicate that ANAC032 is an important transcription factor that positively regulates age-dependent and stress-induced senescence in A. thaliana by modulating reactive oxygen species production.

NAC (NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped cotyledon)) proteins are one of the largest families of plant-specific transcription factors, and this family is present in a wide range of land plants. Here, we have investigated the role of ANAC046 in the regulation of suberin biosynthesis and deposition in Arabidopsis. Subcellular localization and transcriptional activity assays showed that ANAC046 localizes in the nucleus, where it functions as a transcription activator. Analysis of the PANAC046:GUS lines revealed that ANAC046 is mainly expressed in the root endodermis and periderm, and is also induced in leaves by wounding. The transgenic lines overexpressing ANAC046 exhibited defective surfaces on the aerial plant parts compared to the wild-type (WT) as characterized by increased permeability for Toluidine blue stain and greater chlorophyll leaching. Quantitative RT-PCR analysis showed that the expression of suberin biosynthesis genes was significantly higher in the roots and leaves of overexpression lines compared to the WT. The biochemical analysis of leaf cuticular waxes showed that the overexpression lines accumulated 30% more waxes than the WT. Concurrently, overexpression lines also deposited almost twice the amount of suberin content in their roots compared with the WT. Taken together, these results showed that ANAC046 is an important transcription factor that promotes suberin biosynthesis in Arabidopsis thaliana roots.

GATA transcription factors are involved in multiple processes in plant growth and development. Two GATA factors, NITRATE‐INDUCIBLE,CARBON METABOLISM‐INVOLVED (GNC) and CYTOKININ‐RESPONSIVE GATA FACTOR 1 (CGA1, also named GNL), are important regulators in greening, flowering, senescence, and hormone signaling. However, their direct target genes related to these biological processes are poorly characterized. Here, GNC and CGA1 are shown to be transcription activators and by using chromatin immunoprecipitation sequencing (ChIP‐seq), 1475 and 638 genes are identified to be associated with GNC and CGA1 binding, respectively. Enrichment of diverse motifs in the peak binding regions for GNC and CGA1 suggests the possibility that these two transcription factors also interact with other transcription factors and in addition genes coding for DNA‐binding proteins are highly enriched among GNC‐ and CGA1‐associated genes. Despite the fact that these two GATA factors are known to share a large portion of co‐expressed genes, our analysis revealed a low percentage of overlapping binding‐associated genes for these two homologues. This suggests a possible cross‐regulation between these, which is verified using ChIP‐qPCR. The common and specific biological processes regulated by GNC and CGA1 also support this notion. Functional analysis of the binding‐associated genes revealed that those encoding transcription factors, E3 ligase, as well as genes with roles in plant development are highly enriched, indicating that GNC and CGA1 mediate complex genetic networks in regulating different aspects of plant growth and development.

Background High-dimensional data of discrete and skewed nature is commonly encountered in high-throughput sequencing studies. Analyzing the network itself or the interplay between genes in this type of data continues to present many challenges. As data visualization techniques become cumbersome for higher dimensions and unconvincing when there is no clear separation between homogeneous subgroups within the data, cluster analysis provides an intuitive alternative. The aim of applying mixture model-based clustering in this context is to discover groups of co-expressed genes, which can shed light on biological functions and pathways of gene products. Results A mixture of multivariate Poisson-log normal (MPLN) model is developed for clustering of high-throughput transcriptome sequencing data. Parameter estimation is carried out using a Markov chain Monte Carlo expectation-maximization (MCMC-EM) algorithm, and information criteria are used for model selection. Conclusions The mixture of MPLN model is able to fit a wide range of correlation and overdispersion situations, and is suited for modeling multivariate count data from RNA sequencing studies. All scripts used for implementing the method can be found at https://github.com/anjalisilva/MPLNClust. Electronic supplementary material The online version of this article (10.1186/s12859-019-2916-0) contains supplementary material, which is available to authorized users.

Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

Chloroplast biogenesis has been well documented in higher plants, yet the complex methods used to regulate chloroplast activity under fluctuating environmental conditions are not well understood. In rice (Oryza sativa), the CYTOKININ-RESPONSIVE GATA TRANSCRIPTION FACTOR1 (Cga1) shows increased expression following light, nitrogen, and cytokinin treatments, while darkness and gibberellin reduce expression. Strong overexpression of Cga1 produces dark green, semidwarf plants with reduced tillering, whereas RNA interference knockdown results in reduced chlorophyll and increased tillering. Coexpression, microarray, and real-time expression analyses demonstrate a correlation between Cga1 expression and the expression of important nucleus-encoded, chloroplast-localized genes. Constitutive Cga1 overexpression increases both chloroplast biogenesis and starch production but also results in delayed senescence and reduced grain filling. Growing the transgenic lines under different nitrogen regimes indicates potential agricultural applications for Cga1, including manipulation of biomass, chlorophyll/chloroplast content, and harvest index. These results indicate a conserved mechanism by which Cga1 regulates chloroplast development in higher plants.

Here, we present a genome-wide overview of transcriptional circuits in the agriculturally significant crop species maize (Zea mays). We examined transcript abundance data at 50 developmental stages, from embryogenesis to senescence, for 34,876 gene models and classified genes into 24 robust coexpression modules. Modules were strongly associated with tissue types and related biological processes. Sixteen of the 24 modules (67%) have preferential transcript abundance within specific tissues. One-third of modules had an absence of gene expression in specific tissues. Genes within a number of modules also correlated with the developmental age of tissues. Coexpression of genes is likely due to transcriptional control. For a number of modules, key genes involved in transcriptional control have expression profiles that mimic the expression profiles of module genes, although the expression of transcriptional control genes is not unusually representative of module gene expression. Known regulatory motifs are enriched in several modules. Finally, of the 13 network modules with more than 200 genes, three contain genes that are notably clustered (P < 0.05) within the genome. This work, based on a carefully selected set of major tissues representing diverse stages of maize development, demonstrates the remarkable power of transcript-level coexpression networks to identify underlying biological processes and their molecular components.

Anthocyanins are known to have antioxidant activities. Their accumulation can be triggered by many chemical and environmental factors, including reactive oxygen species (ROS). However, the mechanism of ROS-induced anthocyanin accumulation and the role of anthocyanins in the response of Arabidopsis (Arabidopsis thaliana) to different stresses are largely unknown. Here, we study the cross-regulation between ROS and anthocyanin production. Ten Arabidopsis mutants covering the main anthocyanin regulatory and biosynthetic genes are systematically analyzed under ROS-generating stresses. We find that ROS triggers anthocyanin accumulation by up-regulating the anthocyanin late biosynthetic and the corresponding regulatory genes. The anthocyanin-deficient mutants have more endogenous ROS and are more sensitive to ROS-generating stresses while having decreased antioxidant capacity. Supplementation with cyanidin makes them less susceptible to ROS, with increased anthocyanin and reduced ROS accumulation. In contrast, pap1-D, which overaccumulates anthocyanins, shows the opposite responses. Gene expression analysis reveals that photosynthetic capacity is more impaired in anthocyanin-deficient mutants under high-light stress. Expression levels of ROS-scavenging enzyme genes are not correlated with the radical-scavenging activity in different mutants. We conclude that ROS are an important source signal to induce anthocyanin accumulation by up-regulating late biosynthetic and the corresponding regulatory genes and, as a feed-back regulation, anthocyanins modulate the ROS level and the sensitivity to ROS-generating stresses in maintaining photosynthetic capacity.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling., Nitrate stimulates seed germination in many plant species. Here, Yan et al. show that the Arabidopsis transcription factor NIN-like protein 8 is required to stimulate germination in response to nitrate and induces expression of an enzyme involved in ABA catabolism.

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, high light and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under high light stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages.

Anthocyanin production is a characteristic response of flowering plants to unfavourable environmental conditions. The potential roles of flavonoids and anthocyanins in plant growth were investigated by growing Arabidopsis thaliana anthocyanin production mutants (transparent testa) under limiting nitrogen and high light conditions. Inability to produce kaempferol or subsequent intermediate compounds by some transparent testa lines was correlated with less biomass accumulation in mature plants compared with wild-type control plants under all growth conditions tested. However, under both limiting nitrogen and high light chronic stress conditions, mutant lines defective in later steps of the anthocyanin production pathway produced the same or more biomass than wild-type plants. No difference in senescence between transparent testa and wild-type plants was found using chlorophyll catabolism and SAG12 expression measurements, and no mutants were impaired in the ability to remobilize nutrients from the vegetative to reproductive tissues. Moreover, the absence of anthocyanin and/or upstream flavonoids does not affect the ability of plants to respond to limiting nitrogen by reducing photosynthetic capacity. These results support a role for kaempferol and quercetin accumulation in normal plant growth and development. Further, the absence of anthocyanins has no effect on plant growth under the chronic stress conditions tested.

The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.

Little is known about the nature of floral inductive cues in day-neutral plants that are insensitive to photoperiod variations and, therefore, rely on endogenous signals to initiate reproductive growth. The INDETERMINATE1 (ID1) transcription factor is a key regulator of the transition to flowering in day-neutral maize. The ID1 gene is expressed exclusively in developing leaves, where it controls the production or transmission of leaf-derived florigenic signals. Florigen-producing source leaves were compared with mature leaves of late-flowering id1 plants, and metabolite and gene expression differences associated with the floral transition in maize were observed. While id1 mutants have a similar capacity for photosynthesis to wild-type siblings, id1 source leaves show quantitative differences in carbohydrate allocation prior to the floral transition stage, with a marked increase in sucrose and other soluble sugars, accompanied by a decrease in tricarboxylic acid (TCA) cycle organic acids. Importantly, source leaves of autonomous-flowering maize are typified by a higher transitory starch to sucrose ratio and a transcript profile enriched for sucrose synthesis and starch metabolism-related gene function. Finally, similar changes in transitory starch and sucrose are not observed in teosinte, the tropical progenitor of maize that requires short-day photoperiods to induce flowering. Together, these data define a transcript and metabolite signature associated with the autonomous floral transition in temperate maize leaves.

Plants interact with their environment by modifying gene expression patterns. One mechanism for this interaction involves epigenetic modifications that affect a number of aspects of plant growth and development. Thus, the epigenome is highly dynamic in response to environmental cues and developmental changes. Flowering is controlled by a set of genes that are affected by environmental conditions through an alteration in their expression pattern. This ensures the production of flowers even when plants are growing under adverse conditions, and thereby enhances transgenerational seed production. In this review recent findings on the epigenetic changes associated with flowering in Arabidopsis thaliana grown under abiotic stress conditions such as cold, drought, and high salinity are discussed. These epigenetic modifications include DNA methylation, histone modifications, and the production of micro RNAs (miRNAs) that mediate epigenetic modifications. The roles played by the phytohormones abscisic acid (ABA) and auxin in chromatin remodelling are also discussed. It is shown that there is a crucial relationship between the epigenetic modifications associated with floral initiation and development and modifications associated with stress tolerance. This relationship is demonstrated by the common epigenetic pathways through which plants control both flowering and stress tolerance, and can be used to identify new epigenomic players.

The endoplasmic reticulum (ER) is a dynamic organelle that consists of numerous regions or 'subdomains' that have discrete morphological features and functional properties. Although it is generally accepted that these subdomains differ in their protein and perhaps lipid compositions, a clear understanding of how they are assembled and maintained has not been well established. We previously demonstrated that two diacylglycerol acyltransferase enzymes (DGAT1 and DGAT2) from tung tree (Vernicia fordii) were located in different subdomains of ER, but the mechanisms responsible for protein targeting to these subdomains were not elucidated. Here we extend these studies by describing two glycerol-3-phosphate acyltransferase-like (GPAT) enzymes from tung tree, GPAT8 and GPAT9, that both colocalize with DGAT2 in the same ER subdomains. Measurement of protein-protein interactions using the split-ubiquitin assay revealed that GPAT8 interacts with itself, GPAT9 and DGAT2, but not with DGAT1. Furthermore, mutational analysis of GPAT8 revealed that the protein's first predicted hydrophobic region, which contains an amphipathic helix-like motif, is required for interaction with DGAT2 and for DGAT2-dependent colocalization in ER subdomains. Taken together, these results suggest that the regulation and organization of ER subdomains is mediated at least in part by higher-ordered, hydrophobic-domain-dependent homo- and hetero-oligomeric protein-protein interactions.

Cytosolic glutamine synthetase (GS1) is responsible for the primary assimilation of ammonia, and a role in nitrogen (N) remobilization is implicated from its vascular localization and enhanced expression during senescence. This paper tested the hypothesis that overexpression (OX) of GS1 in rice improves utilization N use efficiency (UtE = spikelet yield/shoot N content). Three GS1 OX lines were identified using activity assays and quantitative polymerase chain reaction. Physiological analysis of the OX lines, as well as azygous and wild-type (Wt) controls, was conducted with mature plants after growth under varying nitrate conditions (non-limiting N, limiting N, transfer from non-limiting N to limiting N at panicle emergence) and growth environments (growth chamber vs greenhouse). Overall, OX lines did not differ from azygous controls in vegetative yield or shoot N content. In two of the three growth trials (i.e. the growth chamber trials) harvest index, N harvest index (spikelet N content/shoot N content) and UtE were generally enhanced in the OX lines relative to their azygous controls. These characteristics were highly correlated with percent spikelets filled and spikelet number. Thus, N partitioning in rice during grain filling could be altered by GS1 OX, resulting in improved UtE. Unfortunately, GS OX did not result in more efficient use of N under limiting N than under non-limiting N, and is therefore unlikely to result in the use of less N under field conditions. Transformation effects significantly hindered the productivity of the OX lines, but backcrossing to the Wt should overcome this.

Anthocyanins are water-soluble pigments with antioxidant activities. In plants, multiple factors can trigger the accumulation of anthocyanins, including chemicals and environmental factors. Reactive oxygen species (ROS) are common by-products produced under different biotic and abiotic conditions and cause oxidative stress when accumulated at a high level in plant cells. This in turn leads to the production of anthocyanins. However, the mechanisms of ROS-induced anthocyanin accumulation and the role of anthocyanins in the response of plants to different stresses are largely unknown. We have recently reported the cross-regulation between ROS and anthocyanin production through analyzing ten Arabidopsis mutants covering the main anthocyanin regulatory and biosynthetic genes grown under different ROS-generating stresses. Here, we describe the general phenotypic response of anthocyanin mutants under normal and ROS-generating stress conditions, showing the changing levels of anthocyanin accumulation and their sensitivity to stresses. In addition, we propose a model that describes a particular gene interaction that highlights how the cross-regulation mechanisms between ROS and anthocyanin production are essential for plant resistance to various stresses through removing excessive ROS and maintaining photosynthetic capacity.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

The phytohormone auxin plays a critical role for plant growth by regulating the expression of a set of genes. One large auxin-responsive gene family of this type is the small auxin-up RNA (SAUR) genes, although their function is largely unknown. The expression of the rice (Oryza sativa) SAUR39 gene showed rapid induction by transient change in different environmental factors, including auxin, nitrogen, salinity, cytokinin, and anoxia. Transgenic rice plants overexpressing the SAUR39 gene resulted in lower shoot and root growth, altered shoot morphology, smaller vascular tissue, and lower yield compared with wild-type plants. The SAUR39 gene was expressed at higher levels in older leaves, unlike auxin biosynthesis, which occurs largely in the meristematic region. The transgenic plants had a lower auxin level and a reduced polar auxin transport as well as the down-regulation of some putative auxin biosynthesis and transporter genes. Biochemical analysis also revealed that transgenic plants had lower chlorophyll content, higher levels of anthocyanin, abscisic acid, sugar, and starch, and faster leaf senescence compared with wild-type plants at the vegetative stage. Most of these phenomena have been shown to be negatively correlated with auxin level and transport. Transcript profiling revealed that metabolic perturbations in overexpresser plants were largely due to transcriptional changes of genes involved in photosynthesis, senescence, chlorophyll production, anthocyanin accumulation, sugar synthesis, and transport. The lower growth and yield of overexpresser plants was largely recovered by exogenous auxin application. Taken together, the results suggest that SAUR39 acts as a negative regulator for auxin synthesis and transport.

In Arabidopsis thaliana, the regulation of hexose levels by the large monosaccharide transporter (MST) gene family influences many aspects of plant growth. The cloning and transgenic expression of one family member (STP13) enabled the manipulation of carbon (C) and nitrogen (N) metabolism in Arabidopsis. Transgenic seedlings constitutively over-expressing STP13 (STP13OX) had increased rates of glucose uptake, higher endogenous sucrose levels and accumulated more total C and biomass per plant when grown on soil-less media supplemented with 55 mM glucose and sufficient N (9 mM nitrate). Furthermore, STP13OX seedlings acquired 90% more total N than the Col-0 seedlings, and had higher levels of expression of the nitrate transporter NRT2.2. In addition, STP13OX seedlings were larger and had higher biomass than Col-0 seedlings when grown under a limiting N condition (3 mM nitrate). Transgene analysis of STP13 reveals that its gene product is localized to the plasma membrane (PM) in tobacco BY-2 suspension cells, that it encodes a functional MST in planta, and that the STP13 promoter directs GUS expression to the vasculature and to leaf mesophyll cells. This work highlights the link between C and N metabolism, demonstrating that a plant's N use may be improved by increasing the availability of C.

Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ∼40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

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.

A comprehensive knowledge of mechanisms regulating nitrogen (N) use efficiency is required to reduce excessive input of N fertilizers while maintaining acceptable crop yields under limited N supply. Studying plant species that are naturally adapted to low N conditions could facilitate the identification of novel regulatory genes conferring better N use efficiency. Here, we show that Thellungiella halophila, a halophytic relative of Arabidopsis (Arabidopsis thaliana), grows better than Arabidopsis under moderate (1 mm nitrate) and severe (0.4 mm nitrate) N-limiting conditions. Thellungiella exhibited a lower carbon to N ratio than Arabidopsis under N limitation, which was due to Thellungiella plants possessing higher N content, total amino acids, total soluble protein, and lower starch content compared with Arabidopsis. Furthermore, Thellungiella had higher amounts of several metabolites, such as soluble sugars and organic acids, under N-sufficient conditions (4 mm nitrate). Nitrate reductase activity and NR2 gene expression in Thellungiella displayed less of a reduction in response to N limitation than in Arabidopsis. Thellungiella shoot GS1 expression was more induced by low N than in Arabidopsis, while in roots, Thellungiella GS2 expression was maintained under N limitation but was decreased in Arabidopsis. Up-regulation of NRT2.1 and NRT3.1 expression was higher and repression of NRT1.1 was lower in Thellungiella roots under N-limiting conditions compared with Arabidopsis. Differential transporter gene expression was correlated with higher nitrate influx in Thellungiella at low 15NO3 − supply. Taken together, our results suggest that Thellungiella is tolerant to N-limited conditions and could act as a model system to unravel the mechanisms for low N tolerance.

Nitrogen use efficiency is higher in newer than in older maize (Zea mays L.) hybrids, but the physiological mechanisms underlying differences in N-use efficiency are unknown. The objective of this study was to quantify differences between an older and a newer maize hybrid in their response to N availability throughout the life cycle at both the leaf and the whole-plant level. An older and a newer maize hybrid were grown in a field hydroponic system located near Guelph, ON, in 2005 at a high and a low N level. Leaf carbon exchange rate (CER), chlorophyll index, and the thylakoid electron transport rate (ETR) were measured weekly from 2 wk presilking to 8 wk postsilking. Plant-component dry matter and N content were determined from 1 wk presilking to maturity. At the leaf level, leaf CER declined during the grain-filling period, and the decline was greater under low than high N availability. The decline in leaf CER during the grain-filling period was less in the newer than in the older hybrid under both high and low N availability, and differences in leaf CER were associated most strongly with a reduction in leaf CER per unit absorbed photosynthetic photon flux density. At the whole-plant level, reduction in grain yield in low vs. high N was greater in the older than in the newer hybrid. The hybrid x N interaction for grain yield was attributable predominantly to a greater decline in the proportion of dry matter allocated to the grain in the older hybrid.

Nitrogen is an essential mineral nutrient and is required in great abundance for plant growth and development. Insufficient nitrogen triggers extensive physiological and biochemical changes in plants which constitute a set of adaptive responses to nitrogen limitation. In this study, to determine the genome-wide transcriptome response to nitrogen limitation, Arabidopsis plants were grown with limiting (3 mM) and sufficient (10 mM) nitrate, respectively, and their gene expression profiles were analyzed using Affymetrix GeneChip arrays. In addition to inducing the adaptive responses in Arabidopsis, nitrogen limitation altered the expression levels of 629 genes with 340 up-regulated and 289 down-regulated. The up-regulated group included the genes involved in protein degradation and the biosynthesis of anthocyanin and phenylpropanoids. The down-regulated group contained the genes functioning in photosynthesis and in the synthesis of nitrogenous macromolecules such as chlorophyll, proteins, amino acids and nucleotides. Numerous nitrogen limitation responsive genes encode transcription factors, signal transduction components, and proteins required for hormone synthesis and response. The Arabidopsis nitrogen limitation adaptation mutant (nla) is defective in developing the nitrogen limitation adaptive responses. The microarray analysis revealed that the absence of the functional NLA in the nla mutant extensively altered its responsive transcriptome to nitrogen limitation. In this mutant 1122 genes were up-regulated and 622 repressed. It was also found that the nla mutant phenotype was associated with the early induction of senescence-associated genes. This study presents a genome-wide view of Arabidopsis transcriptome response to nitrogen limitation and its regulation by NLA, and provides information to probe the molecular mechanism controlling plant adaptability to nitrogen limitation.

Over the next 50 years, considerable stress will be placed on worldwide crop production by a combination of factors, including an increased human population, an increase in the crops used per person, and a number of environmental issues. Given current trends, it will be necessary to approximately

Summary Abundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.

Glutaredoxins (GRXs) are small glutathione dependent oxidoreductases that belong to the Thioredoxin (TRX) superfamily and catalyze the reduction of disulfide bonds of their substrate proteins. Plant GRXs include three different groups based on the motif sequence, namely CPYC, CGFS, and CC-type proteins. The rice CC-type proteins, OsGRX6 was identified during the screening for genes whose expression changes depending on the level of available nitrate. Overexpression of OsGRX6 in rice displayed a semi-dwarf phenotype. The OsGRX6 overexpressors contain a higher nitrogen content than the wild type, indicating that OsGRX6 plays a role in homeostatic regulation of nitrogen use. Consistent with this, OsGRX6 overexpressors displayed delayed chlorophyll degradation and senescence compared to the wild type plants. To examine if the growth defect of these transgenic lines attribute to disturbed plant hormone actions, plant hormone levels were measured. The levels of two cytokinins (CKs), 2-isopentenyladenine and trans-zeatin, and gibberellin A1 (GA1) were increased in these lines. We also found that these transgenic lines were less sensitive to exogenously applied GA, suggesting that the increase in GA1 is a result of the feedback regulation. These data suggest that OsGRX6 affects hormone signaling and nitrogen status in rice plants.

In the past 50 years, the application of synthetic nitrogen (N) fertilizer to farmland resulted in a dramatic increase in crop yields but with considerable negative impacts on the environment. New solutions are therefore needed to simultaneously increase yields while maintaining, or preferably decreasing, applied N to maximize the nitrogen use efficiency (NUE) of crops. In this review, we outline the definition of NUE, the selection and development of NUE crops, and the factors that interact with NUE. In particular, we emphasize the challenges of developing crop plants with enhanced NUE, using more classical genetic approaches based on utilizing existing allelic variation for NUE traits. The challenges of phenotyping, mapping quantitative trait loci (QTLs), and selecting candidate genes for NUE improvement are described. In addition, we highlight the importance of different factors that lead to changes in the NUE components of nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE).

Plant microRNAs (miRNAs) are a class of small non-coding RNAs which target and regulate the expression of genes involved in several growth, development, and metabolism processes. Recent researches have shown involvement of miRNAs in the regulation of uptake and utilization of nitrogen (N) and phosphorus (P) and more importantly for plant adaptation to N and P limitation conditions by modifications in plant growth, phenology, and architecture and production of secondary metabolites. Developing strategies that allow for the higher efficiency of using both N and P fertilizers in crop production is important for economic and environmental benefits. Improved crop varieties with better adaptation to N and P limiting conditions could be a key approach to achieve this effectively. Furthermore, understanding on the interactions between N and P uptake and use and their regulation is important for the maintenance of nutrient homeostasis in plants. This review describes the possible functions of different miRNAs and their cross-talk relevant to the plant adaptive responses to N and P limiting conditions. In addition, a comprehensive understanding of these processes at molecular level and importance of biological adaptation for improved N and P use efficiency is discussed.

Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing ∼80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.