Loss of OsMATE6 Function Enhances Drought Resistance Without Yield Penalty by Regulating Stomatal Closure in Rice.

Drought is one of the most severe environmental factors limiting plant growth and crop yield, necessitating the identification of genes that enhance drought resistance for crop improvement. Through screening an ethyl methyl sulfonate-mutagenized rice mutant library, we isolated the PEG tolerance mutant 97-1 (ptm97-1), which displays enhanced resistance to osmotic and drought stress, and increased yield under drought conditions. A point mutation in OsMATE6 was identified as being associated with the drought-resistant phenotype of ptm97-1. The role of OsMATE6 in conferring drought resistance was confirmed by additional OsMATE6 knockout mutants. OsMATE6 is expressed in guard cells, shoots and roots and the OsMATE6-GFP fusion protein predominantly localizes to the plasma membrane. Our ABA efflux assays suggest that OsMATE6 functions as an ABA efflux transporter; mutant protoplasts exhibited a slower ABA release rate compared to the wild type. We hypothesize that OsMATE6 regulates ABA levels in guard cells, influencing stomatal closure and enhancing drought resistance. Notably, OsMATE6 knockout mutants demonstrated greater yields under field drought conditions compared to wild-type plants, highlighting OsMATE6 as a promising candidate for improving crop drought resistance.

The OsNLP3/4-OsRFL module regulates nitrogen-promoted panicle architecture in rice.

Rice panicles, a major component of yield, are regulated by phytohormones and nutrients. How mineral nutrients promote panicle architecture remains largely unknown. Here, we report that NIN-LIKE PROTEIN3 and 4 (OsNLP3/4) are crucial positive regulators of rice panicle architecture in response to nitrogen (N). Loss-of-function mutants of either OsNLP3 or OsNLP4 produced smaller panicles with reduced primary and secondary branches and fewer grains than wild-type, whereas their overexpression plants showed the opposite phenotypes. The OsNLP3/4-regulated panicle architecture was positively correlated with N availability. OsNLP3/4 directly bind to the promoter of OsRFL and activate its expression to promote inflorescence meristem development. Furthermore, OsRFL activates OsMOC1 expression by binding to its promoter. Our findings reveal the novel N-responsive OsNLP3/4-OsRFL-OsMOC1 module that integrates N availability to regulate panicle architecture, shedding light on how N nutrient signals regulate panicle architecture and providing candidate targets for the improvement of crop yield.

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.

Loss of rice PARAQUAT TOLERANCE 3 confers enhanced resistance to abiotic stresses and increases grain yield in field.

Plants frequently suffer from environmental stresses in nature and have evolved sophisticated and efficient mechanisms to cope with the stresses. To balance between growth and stress response, plants are equipped with efficient means to switch off the activated stress responses when stresses diminish. We previously revealed such an off-switch mechanism conferred by Arabidopsis PARAQUAT TOLERANCE 3 (AtPQT3) encoding an E3 ubiquitin ligase, knockout of which significantly enhances resistance to abiotic stresses. To explore whether the rice homologue OsPQT3 is functionally conserved, we generated three knockout mutants with CRISPR-Cas9 technology. The OsPQT3 knockout mutants (ospqt3) display enhanced resistance to oxidative and salt stress with elevated expression of OsGPX1, OsAPX1 and OsSOD1. More importantly, the ospqt3 mutants show significantly enhanced agronomic performance with higher yield compared with the wild type under salt stress in greenhouse as well as in field conditions. We further showed that OsPQT3 expression rapidly decreased in response to oxidative and other abiotic stresses as AtPQT3 does. Taken together, these results show that OsPQT3 is functionally well conserved in rice as an off-switch in stress response as AtPQT3 in Arabidopsis. Therefore, PQT3 locus provides a promising candidate for crop improvement with enhanced stress resistance by gene editing technology.

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.

[Investigation on Spectral Characteristics of Micro-Gap Dielectric Barrier Uniform Discharge in Air].

The plasmas generated by dielectric barrier discharge in atmospheric pressure air have wide application prospect in industry. In order to study generation condition and mechanism, the dielectric barrier uniform discharge in atmospheric pressure air has been studied experimentally with a micro-gap discharge device. Results of electrical characteristics indicate that it exist several current pulses with short width in half period of the applied voltage at a low voltage, a large number of micro-discharge filaments are observed. The discharge power increases with increasing peak value of applied voltage, the micro-discharge filaments increase meanwhile. When the peak of applied voltage reaches to 9.2 kV, only a discharge hump with a width of about 5.5 µs appears in a half period of the applied voltage, micro-discharge filaments cannot be discerned. The uniform discharge has been obtained finally as the micro-discharge filaments extend and superimpose randomly. The emission spectrum of dielectric barrier discharge scanning from 330 to 420 nm is collected. It is found that the intensity of 337.1 nm is stronger than that of 391.4 nm. If the intensity of 337.1 nm is considered as the datum reference, the intensity of 391.4 nm shows the magnitude of electron average energy. The molecule internal energy is evaluated by vibration temperature. The electron average energy and molecule internal energy have been investigated by optical emission spectra. It is found that both of them decrease with increasing the applied voltage. Results indicate that it is not easy to form filamentary discharge when the electrical energy is lower. The average electron energy of uniform discharge is lower than that of the filamentary discharge. These results are of great significance to the application of dielectric barrier uniform discharge obtained in air at atmospheric pressure.

Knockout of OsSPL10 confers enhanced glufosinate resistance in rice.

This study shows that OsSPL10 is a novel genetic locus of glufosinate resistance in rice. OsSPL10 negatively regulates the expression of OsGS genes and thereby decreases GS activity. Knockout of OsSLP10 thus enhances glufosinate resistance, making it a candidate gene for improvement of crop glufosinate and stress resistance.

OsNLP3 enhances grain weight and reduces grain chalkiness in rice.

Grain weight, a key determinant of yield in rice (Oryza sativa L.), is governed primarily by genetic factors, whereas grain chalkiness, a detriment to grain quality, is intertwined with environmental factors such as mineral nutrients. Nitrogen (N) is recognized for its effect on grain chalkiness, but the underlying molecular mechanisms remain to be clarified. This study revealed the pivotal role of rice NODULE INCEPTION-LIKE PROTEIN 3 (OsNLP3) in simultaneously regulating grain weight and grain chalkiness. Our investigation showed that loss of OsNLP3 leads to a reduction in both grain weight and dimension, in contrast to the enhancement observed with OsNLP3 overexpression. OsNLP3 directly suppresses the expression of OsCEP6.1 and OsNF-YA8, which were identified as negative regulators associated with grain weight. Consequently, two novel regulatory modules, OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8, were identified as key players in grain weight regulation. Notably, the OsNLP3-OsNF-YA8 module not only increases grain weight but also mitigates grain chalkiness in response to N. This research clarifies the molecular mechanisms that orchestrate grain weight through the OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8 modules, highlighting the pivotal role of the OsNLP3-OsNF-YA8 module in alleviating grain chalkiness. These findings reveal potential targets for simultaneous enhancement of rice yield and quality.

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.

Nitrate-responsive OsMADS27 promotes salt tolerance in rice.

Salt stress is a major constraint on plant growth and yield. Nitrogen (N) fertilizers are known to alleviate salt stress. However, the underlying molecular mechanisms remain unclear. Here, we show that nitrate-dependent salt tolerance is mediated by OsMADS27 in rice. The expression of OsMADS27 is specifically induced by nitrate. The salt-inducible expression of OsMADS27 is also nitrate dependent. OsMADS27 knockout mutants are more sensitive to salt stress than the wild type, whereas OsMADS27 overexpression lines are more tolerant. Transcriptomic analyses revealed that OsMADS27 upregulates the expression of a number of known stress-responsive genes as well as those involved in ion homeostasis and antioxidation. We demonstrate that OsMADS27 directly binds to the promoters of OsHKT1.1 and OsSPL7 to regulate their expression. Notably, OsMADS27-mediated salt tolerance is nitrate dependent and positively correlated with nitrate concentration. Our results reveal the role of nitrate-responsive OsMADS27 and its downstream target genes in salt tolerance, providing a molecular mechanism for the enhancement of salt tolerance by nitrogen fertilizers in rice. OsMADS27 overexpression increased grain yield under salt stress in the presence of sufficient nitrate, suggesting that OsMADS27 is a promising candidate for the improvement of salt tolerance in rice.

A gain-of-function mutation of the MATE family transporter DTX6 confers paraquat resistance in Arabidopsis.

Paraquat is one of the most widely used nonselective herbicides and has elicited the emergence of paraquat-resistant weeds. However, the molecular mechanisms of paraquat resistance are not completely understood. Here we report the Arabidopsis gain-of-function mutant pqt15-D with significantly enhanced resistance to paraquat and the corresponding gene PQT15, which encodes the Multidrug and Toxic Extrusion (MATE) transporter DTX6. A point mutation at +932 bp in DTX6 causes a G311E amino acid substitution, enhancing the paraquat resistance of pqt15-D, and overexpression of DTX6/PQT15 in the wild-type plants also results in strong paraquat resistance. Moreover, heterologous expression of DTX6 and DTX6-D in Escherichia coli significantly enhances bacterial resistance to paraquat. Importantly, overexpression of DTX6-D enables Arabidopsis plants to tolerate 4 mM paraquat, a near-commercial application level. DTX6/PQT15 is localized in the plasma membrane and endomembrane, and functions as a paraquat efflux transporter as demonstrated by paraquat efflux assays with isolated protoplasts and bacterial cells. Taken together, our results demonstrate that DTX6/PQT15 is an efflux transporter that confers paraquat resistance by exporting paraquat out of the cytosol. These findings reveal a molecular mechanism of paraquat resistance in higher plants and provide a promising candidate gene for engineering paraquat-resistant crops.

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.