Overexpression of ANAC046 Promotes Suberin Biosynthesis in Roots of Arabidopsis thaliana.

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.

Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice.

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.

Overexpression of OsGATA12 regulates chlorophyll content, delays plant senescence and improves rice yield under high density planting.

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.

OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis, transport and distribution.

Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier-like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed-setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) -localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin-related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.

Expression of OsMYB55 in maize activates stress-responsive genes and enhances heat and drought tolerance.

BACKGROUND: Plant response mechanisms to heat and drought stresses have been considered in strategies for generating stress tolerant genotypes, but with limited success. Here, we analyzed the transcriptome and improved tolerance to heat stress and drought of maize plants over-expressing the OsMYB55 gene. RESULTS: Over-expression of OsMYB55 in maize decreased the negative effects of high temperature and drought resulting in improved plant growth and performance under these conditions. This was evidenced by the higher plant biomass and reduced leaf damage exhibited by the transgenic lines compared to wild type when plants were subjected to individual or combined stresses and during or after recovery from stress. A global transcriptomic analysis using RNA sequencing revealed that several genes induced by heat stress in wild type plants are constitutively up-regulated in OsMYB55 transgenic maize. In addition, a significant number of genes up-regulated in OsMYB55 transgenic maize under control or heat treatments have been associated with responses to abiotic stresses including high temperature, dehydration and oxidative stress. The latter is a common and major consequence of imposed heat and drought conditions, suggesting that this altered gene expression may be associated with the improved stress tolerance in these transgenic lines. Functional annotation and enrichment analysis of the transcriptome also pinpoint the relevance of specific biological processes for stress responses. CONCLUSIONS: Our results show that expression of OsMYB55 can improve tolerance to heat stress and drought in maize plants. Enhanced expression of stress-associated genes may be involved in OsMYB55-mediated stress tolerance. Possible implications for the improved tolerance to heat stress and drought of OsMYB55 transgenic maize are discussed.

Ammonium-induced architectural and anatomical changes with altered suberin and lignin levels significantly change water and solute permeabilities of rice (Oryza sativa L.) roots.

MAIN CONCLUSION: Non-optimal ammonium levels significantly alter root architecture, anatomy and root permeabilities for water and nutrient ions. Higher ammonium levels induced strong apoplastic barriers whereas it was opposite for lower levels. Application of nitrogen fertilizer increases crop productivity. However, non-optimal applications can have negative effects on plant growth and development. In this study, we investigated how different levels of ammonium (NH4 (+)) [low (30 or 100 µM) or optimum (300 µM) or high (1000 or 3000 µM)] affect physio-chemical properties of 1-month-old, hydroponically grown rice roots. Different NH4 (+) treatments markedly altered the root architecture and anatomy. Plants grown in low NH4 (+) had the longest roots with a weak deposition of suberised and lignified apoplastic barriers, and it was opposite for plants grown in high NH4 (+). The relative expression levels of selected suberin and lignin biosynthesis candidate genes, determined using qRT-PCR, were lowest in the roots from low NH4 (+), whereas, they were highest for those grown in high NH4 (+). This was reflected by the suberin and lignin contents, and was significantly lower in roots from low NH4 (+) resulting in greater hydraulic conductivity (Lp r) and solute permeability (P sr) than roots from optimum NH4 (+). In contrast, roots grown at high NH4 (+) had markedly greater suberin and lignin contents, which were reflected by strong barriers. These barriers significantly decreased the P sr of roots but failed to reduce the Lp r below those of roots grown in optimum NH4 (+), which can be explained in terms of the physical properties of the molecules used and the size of pores in the apoplast. It is concluded that, in rice, non-optimal NH4 (+) levels differentially affected root properties including Lp r and P sr to successfully adapt to the changing root environment.

High throughput RNA sequencing of a hybrid maize and its parents shows different mechanisms responsive to nitrogen limitation.

BACKGROUND: Development of crop varieties with high nitrogen use efficiency (NUE) is crucial for minimizing N loss, reducing environmental pollution and decreasing input cost. Maize is one of the most important crops cultivated worldwide and its productivity is closely linked to the amount of fertilizer used. A survey of the transcriptomes of shoot and root tissues of a maize hybrid line and its two parental inbred lines grown under sufficient and limiting N conditions by mRNA-Seq has been conducted to have a better understanding of how different maize genotypes respond to N limitation. RESULTS: A different set of genes were found to be N-responsive in the three genotypes. Many biological processes important for N metabolism such as the cellular nitrogen compound metabolic process and the cellular amino acid metabolic process were enriched in the N-responsive gene list from the hybrid shoots but not from the parental lines' shoots. Coupled to this, sugar, carbohydrate, monosaccharide, glucose, and sorbitol transport pathways were all up-regulated in the hybrid, but not in the parents under N limitation. Expression patterns also differed between shoots and roots, such as the up-regulation of the cytokinin degradation pathway in the shoots of the hybrid and down-regulation of that pathway in the roots. The change of gene expression under N limitation in the hybrid resembled the parent with the higher NUE trait. The transcript abundances of alleles derived from each parent were estimated using polymorphic sites in mapped reads in the hybrid. While there were allele abundance differences, there was no correlation between these and the expression differences seen between the hybrid and the two parents. CONCLUSIONS: Gene expression in two parental inbreds and the corresponding hybrid line in response to N limitation was surveyed using the mRNA-Seq technology. The data showed that the three genotypes respond very differently to N-limiting conditions, and the hybrid clearly has a unique expression pattern compared to its parents. Our results expand our current understanding of N responses and will help move us forward towards effective strategies to improve NUE and enhance crop production.

Metabolic and co-expression network-based analyses associated with nitrate response in rice.

BACKGROUND: Understanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions. RESULTS: A gene co-expression network representing clusters of rice genes with similar expression patterns across four nitrogen conditions and two tissue types was generated. The resulting 18 clusters were analyzed for enrichment of significant gene ontology (GO) terms. Four clusters exhibited significant correlation with limiting and reducing nitrate treatments. Among the identified enriched GO terms, those related to nucleoside/nucleotide, purine and ATP binding, defense response, sugar/carbohydrate binding, protein kinase activities, cell-death and cell wall enzymatic activity are enriched. Although a subset of functional categories are more broadly associated with the response of rice organs to limiting N and N reduction, our analyses suggest that N reduction elicits a response distinguishable from that to adaptation to limiting N, particularly in leaves. This observation is further supported by metabolic profiling which shows that several compounds in leaves change proportionally to the nitrate level (i.e. higher in sufficient N vs. limiting N) and respond with even higher levels when the nitrate level is reduced. Notably, these compounds are directly involved in N assimilation, transport, and storage (glutamine, asparagine, glutamate and allantoin) and extend to most amino acids. Based on these data, we hypothesize that plants respond by rapidly mobilizing stored vacuolar nitrate when N deficit is perceived, and that the response likely involves phosphorylation signal cascades and transcriptional regulation. CONCLUSIONS: The co-expression network analysis and metabolic profiling performed in rice pinpoint the relevance of signal transduction components and regulation of N mobilization in response to limiting N conditions and deepen our understanding of N responses and N use in crops.

A developmental transcriptional network for maize defines coexpression modules.

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.

Rice cytokinin GATA transcription Factor1 regulates chloroplast development and plant architecture.

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.

The challenges of commercializing second-generation transgenic crop traits necessitate the development of international public sector research infrastructure.

It has been 30 years since the first transformation of a gene into a plant species, and since that time a number of biotechnology products have been developed, with the most important being insect- and herbicide-resistant crops. The development of second-generation products, including nutrient use efficiency and tolerance to important environmental stressors such as drought, has, up to this time, been less successful. This is in part due to the inherent complexities of these traits and in part due to limitations in research infrastructure necessary for public sector researchers to test their best ideas. Here we discuss lessons from previous work in the generation of the first-generation traits, as well as work from our labs and others on identifying genes for nitrogen use efficiency. We then describe some of the issues that have impeded rapid progress in this area. Finally, we propose the type of public sector organization that we feel is necessary to make advances in important second-generation traits such as nitrogen use efficiency.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

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.

Genome-wide expression profiling of maize in response to individual and combined water and nitrogen stresses.

BACKGROUND: Water and nitrogen are two of the most critical inputs required to achieve the high yield potential of modern corn varieties. Under most agricultural settings however they are often scarce and costly. Fortunately, tremendous progress has been made in the past decades in terms of modeling to assist growers in the decision making process and many tools are now available to achieve more sustainable practices both environmentally and economically. Nevertheless large gaps remain between our empirical knowledge of the physiological changes observed in the field in response to nitrogen and water stresses, and our limited understanding of the molecular processes leading to those changes. RESULTS: This work examines in particular the impact of simultaneous stresses on the transcriptome. In a greenhouse setting, corn plants were grown under tightly controlled nitrogen and water conditions, allowing sampling of various tissues and stress combinations. A microarray profiling experiment was performed using this material and showed that the concomitant presence of nitrogen and water limitation affects gene expression to an extent much larger than anticipated. A clustering analysis also revealed how the interaction between the two stresses shapes the patterns of gene expression over various levels of water stresses and recovery. CONCLUSIONS: Overall, this study suggests that the molecular signature of a specific combination of stresses on the transcriptome might be as unique as the impact of individual stresses, and hence underlines the difficulty to extrapolate conclusions obtained from the study of individual stress responses to more complex settings.

The APETALA-2-like transcription factor OsAP2-39 controls key interactions between abscisic acid and gibberellin in rice.

The interaction between phytohormones is an important mechanism which controls growth and developmental processes in plants. Deciphering these interactions is a crucial step in helping to develop crops with enhanced yield and resistance to environmental stresses. Controlling the expression level of OsAP2-39 which includes an APETALA 2 (AP2) domain leads to phenotypic changes in rice. Overexpression of OsAP2-39 leads to a reduction in yield by decreasing the biomass and the number of seeds in the transgenic rice lines. Global transcriptome analysis of the OsAP2-39 overexpression transgenic rice revealed the upregulation of a key abscisic acid (ABA) biosynthetic gene OsNCED-I which codes for 9-cis-epoxycarotenoid dioxygenase and leads to an increase in the endogenous ABA level. In addition to OsNCED-1, the gene expression analysis revealed the upregulation of a gene that codes for the Elongation of Upper most Internode (EUI) protein, an enzyme that catalyzes 16α, 17-epoxidation of non-13-hydroxylated GAs, which has been shown to deactivate gibberellins (GAs) in rice. The exogenous application of GA restores the wild-type phenotype in the transgenic line and ABA application induces the expression of EUI and suppresses the expression of OsAP2-39 in the wild-type line. These observations clarify the antagonistic relationship between ABA and GA and illustrate a mechanism that leads to homeostasis of these hormones. In vivo and in vitro analysis showed that the expression of both OsNCED-1 and EUI are directly controlled by OsAP2-39. Together, these results reveal a novel mechanism for the control of the ABA/GA balance in rice which is regulated by OsAP2-39 that in turn regulates plant growth and seed production.

Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency.

Development of genetic varieties with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. Generally, NUE can be divided into two parts. First, assimilation efficiency involves nitrogen (N) uptake and assimilation and second utilization efficiency involves N remobilization. Understanding the mechanisms regulating these processes is crucial for the improvement of NUE in crop plants. One important approach is to develop an understanding of the plant response to different N regimes, especially to N limitation, using various methods including transcription profiling, analysing mutants defective in their normal response to N limitation, and studying plants that show better growth under N-limiting conditions. One can then attempt to improve NUE in crop plants using the knowledge gained from these studies. There are several potential genetic and molecular approaches for the improvement of crop NUE discussed in this review. Increased knowledge of how plants respond to different N levels as well as to other environmental conditions is required to achieve this.

Reappraisal of nitrogen use efficiency in rice overexpressing glutamine synthetase1.

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.

Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling.

Development of genetic varieties with improved nitrogen-use efficiency (NUE) is essential for sustainable agriculture. In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified N-responsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops.

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.

Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis.

BACKGROUND: A large quantity of nitrogen (N) fertilizer is used for crop production to achieve high yields at a significant economic and environmental cost. Efforts have been directed to understanding the molecular basis of plant responses to N and identifying N-responsive genes in order to manipulate their expression, thus enabling plants to use N more efficiently. No studies have yet delineated these responses at the transcriptional level when plants are grown under chronic N stress and the understanding of regulatory elements involved in N response is very limited. RESULTS: To further our understanding of the response of plants to varying N levels, a growth system was developed where N was the growth-limiting factor. An Arabidopsis whole genome microarray was used to evaluate global gene expression under different N conditions. Differentially expressed genes under mild or severe chronic N stress were identified. Mild N stress triggered only a small set of genes significantly different at the transcriptional level, which are largely involved in various stress responses. Plant responses were much more pronounced under severe N stress, involving a large number of genes in many different biological processes. Differentially expressed genes were also identified in response to short- and long-term N availability increases. Putative N regulatory elements were determined along with several previously known motifs involved in the responses to N and carbon availability as well as plant stress. CONCLUSION: Differentially expressed genes identified provide additional insights into the coordination of the complex N responses of plants and the components of the N response mechanism. Putative N regulatory elements were identified to reveal possible new components of the regulatory network for plant N responses. A better understanding of the complex regulatory network for plant N responses will help lead to strategies to improve N use efficiency.

Altered Expression of OsNLA1 Modulates Pi Accumulation in Rice (Oryza sativa L.) Plants.

Current agricultural practices rely on heavy use of fertilizers for increased crop productivity. However, the problems associated with heavy fertilizer use, such as high cost and environmental pollution, require the development of crop species with increased nutrient use efficiency. In this study, by using transgenic approaches, we have revealed the critical role of OsNLA1 in phosphate (Pi) accumulation of rice plants. When grown under sufficient Pi and nitrate levels, OsNLA1 knockdown (Osnla1-1, Osnla1-2, and Osnla1-3) lines accumulated higher Pi content in their shoot tissues compared to wild-type, whereas, over-expression lines (OsNLA1-OE1, OsNLA1-OE2, and OsNLA1-OE3) accumulated the least levels of Pi. However, under high Pi levels, knockdown lines accumulated much higher Pi content compared to wild-type and exhibited Pi toxicity symptoms in the leaves. In contrast, the over-expression lines had 50-60% of the Pi content of wild-type and did not show such symptoms. When grown under limiting nitrate levels, OsNLA1 transgenic lines also displayed a similar pattern in Pi accumulation and Pi toxicity symptoms compared to wild-type suggesting an existence of cross-talk between nitrogen (N) and phosphorous (P), which is regulated by OsNLA1. The greater Pi accumulation in knockdown lines was a result of enhanced Pi uptake/permeability of roots compared to the wild-type. The cross-talk between N and P was found to be nitrate specific since the knockdown lines failed to over-accumulate Pi under low (sub-optimal) ammonium level. Moreover, OsNLA1 was also found to interact with OsPHO2, a known regulator of Pi homeostasis, in a Yeast Two-Hybrid (Y2H) assay. Taken together, these results show that OsNLA1 is involved in Pi homeostasis regulating Pi uptake and accumulation in rice plants and may provide an opportunity to enhance P use efficiency by manipulating nitrate supply in the soil.