GAF domain is essential for nitrate-dependent AtNLP7 function.

Nitrate is an essential nutrient and an important signaling molecule in plants. However, the molecular mechanisms by which plants perceive nitrate deficiency signaling are still not well understood. Here we report that AtNLP7 protein transport from the nucleus to the cytoplasm in response to nitrate deficiency is dependent on the N-terminal GAF domain. With the deletion of the GAF domain, AtNLP7ΔGAF always remains in the nucleus regardless of nitrate availability. AtNLP7 ΔGAF also shows reduced activation of nitrate-induced genes due to its impaired binding to the nitrate-responsive cis-element (NRE) as well as decreased growth like nlp7-1 mutant. In addition, AtNLP7ΔGAF is unable to mediate the reduction of reactive oxygen species (ROS) accumulation upon nitrate treatment. Our investigation shows that the GAF domain of AtNLP7 plays a critical role in the sensing of nitrate deficiency signal and in the nitrate-triggered ROS signaling process.

Rice NIN-LIKE PROTEIN 3 modulates nitrogen use efficiency and grain yield under nitrate-sufficient conditions.

Nitrogen (N) is an essential macronutrient for crop growth and yield. Improving the N use efficiency (NUE) of crops is important to agriculture. However, the molecular mechanisms underlying NUE regulation remain largely elusive. Here we report that the OsNLP3 (NIN-like protein 3) regulates NUE and grain yield in rice under N sufficient conditions. OsNLP3 transcript level is significantly induced by N starvation and its protein nucleocytosolic shuttling is specifically regulated by nitrate. Loss-of-function of OsNLP3 reduces plant growth, grain yield, and NUE under sufficient nitrate conditions, whereas under low nitrate or different ammonium conditions, osnlp3 mutants show no clear difference from the wild type. Importantly, under sufficient N conditions in the field, OsNLP3 overexpression lines display improved grain yield and NUE compared with the wild type. OsNLP3 orchestrates the expression of multiple N uptake and assimilation genes by directly binding to the nitrate-responsive cis-elements in their promoters. Overall, our study demonstrates that OsNLP3, together with OsNLP1 and OsNLP4, plays overlapping and differential roles in N acquisition and NUE, and modulates NUE and the grain yield increase promoted by N fertilizer. Therefore, OsNLP3 is a promising candidate gene for the genetic improvement of grain yield and NUE in rice.

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency.

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

HOMEOBOX PROTEIN52 Mediates the Crosstalk between Ethylene and Auxin Signaling during Primary Root Elongation by Modulating Auxin Transport-Related Gene Expression.

The gaseous hormone ethylene participates in many physiological processes in plants. Ethylene-inhibited root elongation involves PIN-FORMED2 (PIN2)-mediated basipetal auxin transport, but the molecular mechanisms underlying the regulation of PIN2 function by ethylene (and therefore auxin distribution) are poorly understood. Here, we report that the plant-specific and ethylene-responsive HD-Zip gene HB52 is involved in ethylene-mediated inhibition of primary root elongation in Arabidopsis thaliana Biochemical and genetic analyses demonstrated that HB52 is ethylene responsive and acts downstream of ETHYLENE-INSENSITIVE3 (EIN3). HB52 knockdown mutants displayed an ethylene-insensitive phenotype during primary root elongation, while its overexpression resulted in short roots, as observed in ethylene-treated plants. In addition, root auxin distribution and gravitropism were impaired in HB52 knockdown and overexpression lines. Consistent with these findings, in vitro and in vivo binding experiments showed that HB52 regulates the expression of auxin transport-related genes, including PIN2, WAVY ROOT GROWTH1 (WAG1), and WAG2 by physically binding to their promoter regions. These findings suggest that HB52 functions in the ethylene-mediated inhibition of root elongation by modulating the expression of auxin transport components downstream of EIN3, revealing a mechanism in which HB52 acts as an important node in the crosstalk between ethylene and auxin signaling during plant growth and development.

ARABIDOPSIS NITRATE REGULATED 1 acts as a negative modulator of seed germination by activating ABI3 expression.

Seed germination is a crucial transition point in plant life and is tightly regulated by environmental conditions through the coordination of two phytohormones, gibberellin and abscisic acid (ABA). To avoid unfavorable conditions, plants have evolved safeguard mechanisms for seed germination. The present contribution reports a novel function of the Arabidopsis MCM1/AGAMOUS/DEFICIENS/SRF(MADS)-box transcription factor ARABIDOPSIS NITRATE REGULATED 1 (ANR1) in seed germination. ANR1 knockout mutant is insensitive to ABA, salt and osmotic stress during the seed germination and early seedling development stages, whereas ANR1-overexpressing lines are hypersensitive. ANR1 is responsive to ABA and abiotic stresses and upregulates the expression of ABA Intolerant (ABI)3 to suppress seed germination. ANR1 and ABI3 have similar expression pattern during seed germination. Genetically, ABI3 acts downstream of ANR1. Chromatin immunoprecipitation and yeast-one-hybrid assays showed that ANR1 could bind to the ABI3 promoter to regulate its expression. In addition, ANR1 acts synergistically with AGL21 to suppress seed germination in response to ABA as evidenced by anr1 agl21 double mutant. Taken together, the results herein demonstrate that the ANR1 plays an important role in regulating seed germination and early postgermination growth. ANR1 and AGL21 together constitutes a safeguard mechanism for seed germination to avoid unfavorable conditions.

Rice NIN-LIKE PROTEIN 1 rapidly responds to nitrogen deficiency and improves yield and nitrogen use efficiency.

Nitrogen (N) is indispensable for crop growth and yield, but excessive agricultural application of nitrogenous fertilizers has generated severe environmental problems. A desirable and economical solution to cope with these issues is to improve crop nitrogen use efficiency (NUE). Plant NUE has been a focal point of intensive research worldwide, yet much still has to be learned about its genetic determinants and regulation. Here, we show that rice (Oryza sativa L.) NIN-LIKE PROTEIN 1 (OsNLP1) plays a fundamental role in N utilization. OsNLP1 protein localizes in the nucleus and its transcript level is rapidly induced by N starvation. Overexpression of OsNLP1 improves plant growth, grain yield, and NUE under different N conditions, while knockout of OsNLP1 impairs grain yield and NUE under N-limiting conditions. OsNLP1 regulates nitrate and ammonium utilization by cooperatively orchestrating multiple N uptake and assimilation genes. Chromatin immunoprecipitation and yeast one-hybrid assays showed that OsNLP1 can directly bind to the promoter of these genes to activate their expression. Therefore, our results demonstrate that OsNLP1 is a key regulator of N utilization and represents a potential target for improving NUE and yield in rice.

Arabidopsis ERF1 Mediates Cross-Talk between Ethylene and Auxin Biosynthesis during Primary Root Elongation by Regulating ASA1 Expression.

The gaseous phytohormone ethylene participates in the regulation of root growth and development in Arabidopsis. It is known that root growth inhibition by ethylene involves auxin, which is partially mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE α1 (WEI2/ASA1), encoding a rate-limiting enzyme in tryptophan (Trp) biosynthesis, from which auxin is derived. However, the molecular mechanism by which ethylene decreases root growth via ASA1 is not understood. Here we report that the ethylene-responsive AP2 transcription factor, ETHYLENE RESPONSE FACTOR1 (ERF1), plays an important role in primary root elongation of Arabidopsis. Using loss- and gain-of-function transgenic lines as well as biochemical analysis, we demonstrate that ERF1 can directly up-regulate ASA1 by binding to its promoter, leading to auxin accumulation and ethylene-induced inhibition of root growth. This discloses one mechanism linking ethylene signaling and auxin biosynthesis in Arabidopsis roots.

Correction: Arabidopsis ERF1 Mediates Cross-Talk between Ethylene and Auxin Biosynthesis during Primary Root Elongation by Regulating ASA1 Expression.

[This corrects the article DOI: 10.1371/journal.pgen.1005760.].

Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field.

Drought and salinity are two major environmental factors limiting crop production worldwide. Improvement of drought and salt tolerance of crops with transgenic approach is an effective strategy to meet the demand of the ever-growing world population. Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a homeodomain-START transcription factor, has been demonstrated to significantly improve drought tolerance in Arabidopsis, tobacco, tall fescue and rice. Here we report that AtHDG11 also confers drought and salt tolerance in upland cotton (Gossypium hirsutum) and woody plant poplar (Populus tomentosa Carr.). Our results showed that both the transgenic cotton and poplar exhibited significantly enhanced tolerance to drought and salt stress with well-developed root system. In the leaves of the transgenic cotton plants, proline content, soluble sugar content and activities of reactive oxygen species-scavenging enzymes were significantly increased after drought and salt stress compared with wild type. Leaf stomatal density was significantly reduced, whereas stomatal and leaf epidermal cell size were significantly increased in both the transgenic cotton and poplar plants. More importantly, the transgenic cotton showed significantly improved drought tolerance and better agronomic performance with higher cotton yield in the field both under normal and drought conditions. These results demonstrate that AtHDG11 is not only a promising candidate for crops improvement but also for woody plants.

Activation of 5-HT(2C) receptor promotes the expression of neprilysin in U251 human glioma cells.

Abeta accumulation, a hallmark of Alzheimer's disease, promotes the disease progress in multiple facets. Abeta is formed through amyloidogenic cleavage pathway of amyloid precursor protein (APP). Production of Abeta can be decreased via activation of 5-HT2C receptor, which enhances alternative APP non-amyloidogenic cleavage. Besides, as one of the best characterized Aß degrading enzymes, neprilysin (NEP) in AD progress has drawn more and more attention. We investigated whether there exists any connection between 5-HT2C receptor and NEP expression. The mRNA and protein expressions of NEP were increased after treatment of 5-HT2C receptor agonist RO-60-0175 in concentration- and time-dependent manners, and NEP expression was decreased after treatment of 5-HT2C receptor antagonist SB242084 correspondingly. These results suggest that 5-HT2C receptor may inhibit the Abeta formation by promoting NEP expression. The underlying mechanism will be explored in follow-up study and may provide potential target for AD therapy.

Migration of phosphorus in pig manure during pyrolysis process and slow-release mechanism of biochar in hydroponic application.

Pyrolysis is an effective method for treating of livestock and poultry manure developed in recent years. It can completely decompose pathogens and antibiotics, stabilize heavy metals, and enrich phosphorus (P) in biochar. To elucidate the P migration mechanism under different pig manure pyrolysis temperatures, sequential fractionation, solution 31P nuclear magnetic resonance, X-ray photoelectron spectroscopy, X-ray diffraction, and K-edge X-ray absorption near-edge structure techniques were used to analyze the P species in pig manure biochar (PMB). The results indicated that most of the organic P in the pig manure was converted to inorganic P during pyrolysis. Moreover, the transformation to different P groups pathways was clarified. The phase transition from amorphous to crystalline calcium phosphate was promoted when the temperature was above 600 °C. The content of P extracted by hydrochloric acid, which was the long-term available P for plant uptake, increased significantly. PMB pyrolyzed at 600 °C can be used as a highly effective substitute for P source. It provides the necessary P species (e.g. water-soluble P.) and metal elements for the growth of water spinach plants, and which are slow-release comparing with the Hogland nutrient solution.

Balanced nitrogen-iron sufficiency boosts grain yield and nitrogen use efficiency by promoting tillering.

Crop yield plays a critical role in global food security. For optimal plant growth and maximal crop yields, nutrients must be balanced. However, the potential significance of balanced nitrogen-iron (N-Fe) for improving crop yield and nitrogen use efficiency (NUE) has not previously been addressed. Here, we show that balanced N-Fe sufficiency significantly increases tiller number and boosts yield and NUE in rice and wheat. NIN-like protein 4 (OsNLP4) plays a pivotal role in maintaining the N-Fe balance by coordinately regulating the expression of multiple genes involved in N and Fe metabolism and signaling. OsNLP4 also suppresses OsD3 expression and strigolactone (SL) signaling, thereby promoting tillering. Balanced N-Fe sufficiency promotes the nuclear localization of OsNLP4 by reducing H2O2 levels, reinforcing the functions of OsNLP4. Interestingly, we found that OsNLP4 upregulates the expression of a set of H2O2-scavenging genes to promote its own accumulation in the nucleus. Furthermore, we demonstrated that foliar spraying of balanced N-Fe fertilizer at the tillering stage can effectively increase tiller number, yield, and NUE of both rice and wheat in the field. Collectively, these findings reveal the previously unrecognized effects of N-Fe balance on grain yield and NUE as well as the molecular mechanism by which the OsNLP4-OsD3 module integrates N-Fe nutrient signals to downregulate SL signaling and thereby promote rice tillering. Our study sheds light on how N-Fe nutrient signals modulate rice tillering and provide potential innovative approaches that improve crop yield with reduced N fertilizer input for benefitting sustainable agriculture worldwide.

ATHB17 enhances stress tolerance by coordinating photosynthesis associated nuclear gene and ATSIG5 expression in response to abiotic stress.

Photosynthesis is sensitive to environmental stress and must be efficiently modulated in response to abiotic stress. However, the underlying mechanisms are not well understood. Here we report that ARABIDOPSIS THALIANA HOMEOBOX 17 (ATHB17), an Arabidopsis HD-Zip transcription factor, regulated the expression of a number of photosynthesis associated nuclear genes (PhANGs) involved in the light reaction and ATSIG5 in response to abiotic stress. ATHB17 was responsive to ABA and multiple stress treatments. ATHB17-overexpressing plants displayed enhanced stress tolerance, whereas its knockout mutant was more sensitive compared to the wild type. Through RNA-seq and quantitative real-time reverse transcription PCR (qRT-PCR) analysis, we found that ATHB17 did not affect the expression of many known stress-responsive marker genes. Interestingly, we found that ATHB17 down-regulated many PhANGs and could directly modulate the expression of several PhANGs by binding to their promoters. Moreover, we identified ATSIG5, encoding a plastid sigma factor, as one of the target genes of ATHB17. Loss of ATSIG5 reduced salt tolerance while overexpression of ATSIG5 enhanced salt tolerance, similar to that of ATHB17. ATHB17 can positively modulate the expression of many plastid encoded genes (PEGs) through regulation of ATSIG5. Taken together, our results suggest that ATHB17 may play an important role in protecting plants by adjusting expression of PhANGs and PEGs in response to abiotic stresses.

Arabidopsis MADS-Box Transcription Factor AGL21 Acts as Environmental Surveillance of Seed Germination by Regulating ABI5 Expression.

Seed germination is a crucial checkpoint for plant survival under unfavorable environmental conditions. Abscisic acid (ABA) signaling plays a vital role in integrating environmental information to regulate seed germination. It has been well known that MCM1/AGAMOUS/DEFICIENS/SRF (MADS)-box transcription factors are key regulators of seed and flower development in Arabidopsis. However, little is known about their functions in seed germination. Here we report that MADS-box transcription factor AGL21 is a negative regulator of seed germination and post-germination growth by controlling the expression of ABA-INSENSITIVE 5 (ABI5) in Arabidopsis. The AGL21-overexpressing plants were hypersensitive to ABA, salt, and osmotic stresses during seed germination and early post-germination growth, whereas agl21 mutants were less sensitive. We found that AGL21 positively regulated ABI5 expression in seeds. Consistently, genetic analyses showed that AGL21 is epistatic to ABI5 in controlling seed germination. Chromatin immunoprecipitation assays further demonstrated that AGL21 could directly bind to the ABI5 promoter in plant cells. Moreover, we found that AGL21 responded to multiple environmental stresses and plant hormones during seed germination. Taken together, our results suggest that AGL21 acts as a surveillance integrator that incorporates environmental cues and endogenous hormonal signals into ABA signaling to regulate seed germination and early post-germination growth.

Overexpression of Arabidopsis NLP7 improves plant growth under both nitrogen-limiting and -sufficient conditions by enhancing nitrogen and carbon assimilation.

Nitrogen is essential for plant survival and growth. Excessive application of nitrogenous fertilizer has generated serious environment pollution and increased production cost in agriculture. To deal with this problem, tremendous efforts have been invested worldwide to increase the nitrogen use ability of crops. However, only limited success has been achieved to date. Here we report that NLP7 (NIN-LIKE PROTEIN 7) is a potential candidate to improve plant nitrogen use ability. When overexpressed in Arabidopsis, NLP7 increases plant biomass under both nitrogen-poor and -rich conditions with better-developed root system and reduced shoot/root ratio. NLP7-overexpressing plants show a significant increase in key nitrogen metabolites, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signalling. More importantly, overexpression of NLP7 also enhances photosynthesis rate and carbon assimilation, whereas knockout of NLP7 impaired both nitrogen and carbon assimilation. In addition, NLP7 improves plant growth and nitrogen use in transgenic tobacco (Nicotiana tabacum). Our results demonstrate that NLP7 significantly improves plant growth under both nitrogen-poor and -rich conditions by coordinately enhancing nitrogen and carbon assimilation and sheds light on crop improvement.

Arabidopsis ERF109 mediates cross-talk between jasmonic acid and auxin biosynthesis during lateral root formation.

Jasmonic acid (JA) is well known to promote lateral root formation but the mechanisms by which JA signalling is integrated into the pathways responsible for lateral root formation, and how it interacts with auxin in this process remains poorly understood. Here, we report that the highly JA-responsive ethylene response factor 109 (ERF109) mediates cross-talk between JA signalling and auxin biosynthesis to regulate lateral root formation in Arabidopsis. erf109 mutants have fewer lateral roots under MeJA treatments compared with wild type whereas ERF109 overexpression causes a root phenotype that resembles those of auxin overproduction mutants. ERF109 binds directly to GCC-boxes in the promoters of ASA1 and YUC2, which encode two key enzymes in auxin biosynthesis. Thus, our study reveals a molecular mechanism for JA and auxin cross-talk during JA-induced lateral root formation.

MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals.

Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth.