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 impact on grain chalkiness, yet the underlying molecular mechanisms remain elusive. 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 the 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 augments grain weight but also mitigates grain chalkiness in response to N. This research clarifies the molecular mechanisms orchestrating grain weight through the OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8 modules, underscoring the pivotal role of the OsNLP3-OsNF-YA8 module in alleviating grain chalkiness. These findings offer potential targets for concurrently enhancing rice yield and quality.

Root-derived long-distance signals trigger ABA synthesis and enhance drought resistance in Arabidopsis.

Vascular plants have evolved intricate long-distance signaling mechanisms to cope with environmental stress, with reactive oxygen species (ROS) emerging as pivotal systemic signals in plant stress responses. However, the exact role of ROS as root-to-shoot signals in the drought response has not been determined. In this study, we reveal that compared with wild-type plants, ferric reductase defective 3 (frd3) mutants exhibit enhanced drought resistance concomitant with elevated NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) transcript levels and abscisic acid (ABA) contents in leaves as well as increased hydrogen peroxide (H2O2) levels in roots and leaves. Grafting experiments distinctly illustrate that drought resistance can be conferred by the frd3 rootstock regardless of the scion genotype, indicating that long-distance signals originating from frd3 roots promote an increase in ABA levels in leaves. Intriguingly, the drought resistance conferred by the frd3 mutant rootstock is weakened by the CAT2-overexpressing scion, suggesting that H2O2 may be involved in long-distance signaling. Moreover, the results of comparative transcriptome and proteome analyses support the drought resistance phenotype of the frd3 mutant. Taken together, our findings substantiate the notion that frd3 root-derived long-distance signals trigger ABA synthesis in leaves and enhance drought resistance, providing new evidence for root-to-shoot long-distance signaling in the drought response of plants.

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.

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.

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.

SUE4, a novel PIN1-interacting membrane protein, regulates acropetal auxin transport in response to sulfur deficiency.

Sulfur (S) is an essential macronutrient for plants and a signaling molecule in abiotic stress responses. It is known that S availability modulates root system architecture; however, the underlying molecular mechanisms are largely unknown. We previously reported an Arabidopsis gain-of-function mutant sulfate utilization efficiency4 (sue4) that could tolerate S deficiency during germination and early seedling growth with faster primary root elongation. Here, we report that SUE4, a novel plasma membrane-localized protein, interacts with the polar auxin transporter PIN1, resulting in reduced PIN1 protein levels and thus decreasing auxin transport to the root tips, which promotes primary root elongation. Moreover, SUE4 is induced by sulfate deficiency, consistent with its role in root elongation. Further analyses showed that the SUE4-PIN1 interaction decreased PIN1 levels, possibly through 26 S proteasome-mediated degradation. Taken together, our finding of SUE4-mediated root elongation is consistent with root adaptation to highly mobile sulfate in soil, thus revealing a novel component in the adaptive response of roots to S deficiency.

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.

14 C-paraquat Efflux Assay in Arabidopsis Mesophyll Protoplasts.

Weeds compete with crops for growth resources, causing tremendous yield losses. Paraquat is one of the three most common non-selective herbicides. To study the mechanisms of paraquat resistance, we need to trace the movement of paraquat in plants and within the cell. 14 C is a radioactive carbon isotope widely used to trace substances of interest in various biological studies, especially in transport analyses. Here, we describe a detailed protocol using 14 C-paraquat to demonstrate paraquat efflux in Arabidopsis protoplasts.

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.

Understanding paraquat resistance mechanisms in Arabidopsis thaliana to facilitate the development of paraquat-resistant crops.

Paraquat (PQ) is the third most used broad-spectrum nonselective herbicide around the globe after glyphosate and glufosinate. Repeated usage and overreliance on this herbicide have resulted in the emergence of PQ-resistant weeds that are a potential hazard to agriculture. It is generally believed that PQ resistance in weeds is due to increased sequestration of the herbicide and its decreased translocation to the target site, as well as an enhanced ability to scavenge reactive oxygen species. However, little is known about the genetic bases and molecular mechanisms of PQ resistance in weeds, and hence no PQ-resistant crops have been developed to date. Forward genetics of the model plant Arabidopsis thaliana has advanced our understanding of the molecular mechanisms of PQ resistance. This review focuses on PQ resistance loci and resistance mechanisms revealed in Arabidopsis and examines the possibility of developing PQ-resistant crops using the elucidated mechanisms.

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.

Arabidopsis MADS-box factor AGL16 is a negative regulator of plant response to salt stress by downregulating salt-responsive genes.

Sessile plants constantly experience environmental stresses in nature. They must have evolved effective mechanisms to balance growth with stress response. Here we report the MADS-box transcription factor AGL16 acting as a negative regulator in stress response in Arabidopsis. Loss-of-AGL16 confers resistance to salt stress in seed germination, root elongation and soil-grown plants, while elevated AGL16 expression confers the opposite phenotypes compared with wild-type. However, the sensitivity to abscisic acid (ABA) in seed germination is inversely correlated with AGL16 expression levels. Transcriptomic comparison revealed that the improved salt resistance of agl16 mutants was largely attributed to enhanced expression of stress-responsive transcriptional factors and the genes involved in ABA signalling and ion homeostasis. We further demonstrated that AGL16 directly binds to the CArG motifs in the promoter of HKT1;1, HsfA6a and MYB102 and represses their expression. Genetic analyses with double mutants also support that HsfA6a and MYB102 are target genes of AGL16. Taken together, our results show that AGL16 acts as a negative regulator transcriptionally suppressing key components in the stress response and may play a role in balancing stress response with growth.

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.

HBI transcription factor-mediated ROS homeostasis regulates nitrate signal transduction.

Nitrate is both an important nutrient and a critical signaling molecule that regulates plant metabolism, growth, and development. Although several components of the nitrate signaling pathway have been identified, the molecular mechanism of nitrate signaling remains unclear. Here, we showed that the growth-related transcription factors HOMOLOG OF BRASSINOSTEROID ENHANCED EXPRESSION2 INTERACTING WITH IBH1 (HBI1) and its three closest homologs (HBIs) positively regulate nitrate signaling in Arabidopsis thaliana. HBI1 is rapidly induced by nitrate through NLP6 and NLP7, which are master regulators of nitrate signaling. Mutations in HBIs result in the reduced effects of nitrate on plant growth and ∼22% nitrate-responsive genes no longer to be regulated by nitrate. HBIs increase the expression levels of a set of antioxidant genes to reduce the accumulation of reactive oxygen species (ROS) in plants. Nitrate treatment induces the nuclear localization of NLP7, whereas such promoting effects of nitrate are significantly impaired in the hbi-q and cat2 cat3 mutants, which accumulate high levels of H2O2. These results demonstrate that HBI-mediated ROS homeostasis regulates nitrate signal transduction through modulating the nucleocytoplasmic shuttling of NLP7. Overall, our findings reveal that nitrate treatment reduces the accumulation of H2O2, and H2O2 inhibits nitrate signaling, thereby forming a feedback regulatory loop to regulate plant growth and development.

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.

HBI1-TCP20 interaction positively regulates the CEPs-mediated systemic nitrate acquisition.

Nitrate is the main source of nitrogen for plants but often distributed heterogeneously in soil. Plants have evolved sophisticated strategies to achieve adequate nitrate by modulating the root system architecture. The nitrate acquisition system is triggered by the short mobile peptides C-TERMINALLY ENCODED PEPTIDES (CEPs) that are synthesized on the nitrate-starved roots, but induce the expression of nitrate transporters on the other nitrate-rich roots through an unclear signal transduction pathway. Here, we demonstrate that the transcription factors HBI1 and TCP20 play important roles in plant growth and development in response to fluctuating nitrate supply. HBI1 physically interacts with TCP20, and this interaction was enhanced by the nitrate starvation. HBI1 and TCP20 directly bind to the promoters of CEPs and cooperatively induce their expression. Mutation in HBIs and/or TCP20 resulted in impaired systemic nitrate acquisition response. Our solid genetic and molecular evidence strongly indicate that the HBI1-TCP20 module positively regulates the CEPs-mediated systemic nitrate acquisition.