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.

The ABI3-ERF1 module mediates ABA-auxin crosstalk to regulate lateral root emergence.

Abscisic acid (ABA) is involved in lateral root (LR) development, but how ABA signaling interacts with auxin signaling to regulate LR formation is not well understood. Here, we report that ABA-responsive ERF1 mediates the crosstalk between ABA and auxin signaling to regulate Arabidopsis LR emergence. ABI3 is a negative factor in LR emergence and transcriptionally activates ERF1 by binding to its promoter, and reciprocally, ERF1 activates ABI3, which forms a regulatory loop that enables rapid signal amplification. Notably, ABI3 physically interacts with ERF1, reducing the cis element-binding activities of both ERF1 and ABI3 and thus attenuating the expression of ERF1-/ABI3-regulated genes involved in LR emergence and ABA signaling, such as PIN1, AUX1, ARF7, and ABI5, which may provide a molecular rheostat to avoid overamplification of auxin and ABA signaling. Taken together, our findings identify the role of the ABI3-ERF1 module in mediating crosstalk between ABA and auxin signaling in LR emergence.

ERF1 inhibits lateral root emergence by promoting local auxin accumulation and repressing ARF7 expression.

Lateral roots (LRs) are crucial for plants to sense environmental signals in addition to water and nutrient absorption. Auxin is key for LR formation, but the underlying mechanisms are not fully understood. Here, we report that Arabidopsis ERF1 inhibits LR emergence by promoting local auxin accumulation with altered distribution and regulating auxin signaling. Loss of ERF1 increases LR density compared with the wild type, whereas ERF1 overexpression causes the opposite phenotype. ERF1 enhances auxin transport by upregulating PIN1 and AUX1, resulting in excessive auxin accumulation in the endodermal, cortical, and epidermal cells surrounding LR primordia. Furthermore, ERF1 represses ARF7 transcription, thereby downregulating the expression of cell-wall remodeling genes that facilitate LR emergence. Together, our study reveals that ERF1 integrates environmental signals to promote local auxin accumulation with altered distribution and repress ARF7, consequently inhibiting LR emergence in adaptation to fluctuating environments.

Transcription elongation factor AtSPT4-2 positively modulates salt tolerance in Arabidopsis thaliana.

BACKGROUND: Salt stress significantly influences plant growth and reduces crop yield. It is highly anticipated to develop salt-tolerant crops with salt tolerance genes and transgenic technology. Hence, it is critical to identify salt tolerance genes that can be used to improve crop salt tolerance. RESULTS: We report that the transcription elongation factor suppressor of Ty 4-2 (SPT4-2) is a positive modulator of salt tolerance in Arabidopsis thaliana. AtSPT4-2 expression is induced by salt stress. Knockout mutants of AtSPT4-2 display a salt-sensitive phenotype, whereas AtSPT4-2 overexpression lines exhibit enhanced salt tolerance. Comparative transcriptomic analyses revealed that AtSPT4-2 may orchestrate the expression of genes associated with salt tolerance, including stress-responsive markers, protein kinases and phosphatases, salt-responsive transcription factors and those maintaining ion homeostasis, suggesting that AtSPT4-2 improves salt tolerance mainly by maintaining ion homeostasis and enhancing stress tolerance. CONCLUSIONS: AtSPT4-2 positively modulates salt tolerance by maintaining ion homeostasis and regulating stress-responsive genes and serves as a candidate for the improvement of crop salt tolerance.

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.

Sulfate-TOR signaling controls transcriptional reprogramming for shoot apex activation.

Plants play a primary role for the global sulfur cycle in the earth ecosystems by reduction of inorganic sulfate from the soil to organic sulfur-containing compounds. How plants sense and transduce the sulfate availability to mediate their growth remains largely unclear. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator of nutrient sensing and metabolic signaling to control cell proliferation and growth in all eukaryotes. By tissue-specific Western blotting and RNA-sequencing analysis, we investigated sulfate-TOR signal pathway in regulating shoot apex development. Here, we report that inorganic sulfate exhibits high potency activating TOR and cell proliferation to promote true leaf development in Arabidopsis in a glucose-energy parallel pathway. Genetic and metabolite analyses suggest that this sulfate activation of TOR is independent from the sulfate-assimilation process and glucose-energy signaling. Significantly, tissue specific transcriptome analyses uncover previously unknown sulfate-orchestrating genes involved in DNA replication, cell proliferation and various secondary metabolism pathways, which largely depends on TOR signaling. Systematic comparison between the sulfate- and glucose-TOR controlled transcriptome further reveals that TOR kinase, as the central growth integrator, responds to different nutrient signals to control both shared and unique transcriptome networks, therefore, precisely modulates plant proliferation, growth and stress responses.

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.

Structure of the Arabidopsis guard cell anion channel SLAC1 suggests activation mechanism by phosphorylation.

Stomata play a critical role in the regulation of gas exchange and photosynthesis in plants. Stomatal closure participates in multiple stress responses, and is regulated by a complex network including abscisic acid (ABA) signaling and ion-flux-induced turgor changes. The slow-type anion channel SLAC1 has been identified to be a central controller of stomatal closure and phosphoactivated by several kinases. Here, we report the structure of SLAC1 in Arabidopsis thaliana (AtSLAC1) in an inactivated, closed state. The cytosolic amino (N)-terminus and carboxyl (C)-terminus of AtSLAC1 are partially resolved and form a plug-like structure which packs against the transmembrane domain (TMD). Breaking the interactions between the cytosolic plug and transmembrane domain triggers channel activation. An inhibition-release model is proposed for SLAC1 activation by phosphorylation that the cytosolic plug dissociates from the transmembrane domain upon phosphorylation, and induces conformational changes to open the pore. These findings facilitate our understanding of the regulation of SLAC1 activity and stomatal aperture in plants.

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.