Loss of OsSPL8 Function Confers Improved Resistance to Glufosinate and Abiotic Stresses in Rice.

Weeds are among the most significant factors contributing to decreases in crop yield and quality. Glufosinate, a nonselective, broad-spectrum herbicide, has been extensively utilized for weed control in recent decades. However, crops are usually sensitive to glufosinate. Therefore, the development of glufosinate-resistant crops is crucial for effective weed management in agriculture. In this study, we characterized a SQUAMOSA promoter-binding-like (SPL) factor, OsSPL8, which acts as a negative regulator of glufosinate resistance by inhibiting the transcription of OsGS1;1 and OsGS2 and consequently reducing GS activity. Furthermore, the loss of OsSPL8 function enhanced tolerance to drought and salt stresses. Transcriptomic comparisons between the gar18-3 mutant and wild type revealed that OsSPL8 largely downregulates stress-responsive genes and upregulates growth-related genes. We demonstrated that OsSPL8 directly regulates OsOMTN6 and OsNAC17, which influence drought tolerance. In addition, OsSPL8 directly represses the expression of salt stress tolerance-related genes such as OsHKT1.1 and OsTPP1. Collectively, our results demonstrate that OsSPL8 is a negative regulator of both glufosinate resistance and abiotic stress tolerance.

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.

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.

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.

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.

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.

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.

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.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

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.

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.

SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response.

Plants are major sulfur reducers in the global sulfur cycle. Sulfate, the major natural sulfur source in soil, is absorbed by plant roots and transported into plastids, where it is reduced and assimilated into Cys for further metabolic processes. Despite its importance, how sulfate is transported into plastids is poorly understood. We previously demonstrated using single Arabidopsis (Arabidopsis thaliana) genetic mutants that each member of the sulfate transporter (SULTR) subfamily 3 was able to transport sulfate across the chloroplast envelope membrane. To resolve the function of SULTR3s, we constructed a sultr3 quintuple mutant completely knocking out all five members of the subfamily. Here we report that all members of the SULTR3 subfamily show chloroplast membrane localization. Sulfate uptake by chloroplasts of the quintuple mutant is reduced by more than 50% compared with the wild type. Consequently, Cys and abscisic acid (ABA) content are reduced to ∼67 and ∼20% of the wild-type level, respectively, and strong positive correlations are found among sulfate, Cys, and ABA content. The sultr3 quintuple mutant shows obvious growth retardation with smaller rosettes and shorter roots. Seed germination of the sultr3 quintuple mutant is hypersensitive to exogenous ABA and salt stress, but is rescued by sulfide supplementation. Furthermore, sulfate-induced stomatal closure is abolished in the quintuple mutant, strongly suggesting that chloroplast sulfate is required for stomatal closure. Our genetic analyses unequivocally demonstrate that sulfate transporter subfamily 3 is responsible for more than half of the chloroplast sulfate uptake and influences downstream sulfate assimilation and ABA biosynthesis.

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.

Arabidopsis MADS-box factor AGL16 negatively regulates drought resistance via stomatal density and stomatal movement.

Drought is one of the most important environmental factors limiting plant growth and productivity. The molecular mechanisms underlying plant drought resistance are complex and not yet fully understood. Here, we show that the Arabidopsis MADS-box transcription factor AGL16 acts as a negative regulator in drought resistance by regulating stomatal density and movement. Loss-of-AGL16 mutants were more resistant to drought stress and had higher relative water content, which was attributed to lower leaf stomatal density and more sensitive stomatal closure due to higher leaf ABA levels compared with the wild type. AGL16-overexpressing lines displayed the opposite phenotypes. AGL16 is preferentially expressed in guard cells and down-regulated in response to drought stress. The expression of CYP707A3 and AAO3 in ABA metabolism and SDD1 in stomatal development was altered in agl16 and overexpression lines, making them potential targets of AGL16. Using chromatin immunoprecipitation, transient transactivation, yeast one-hybrid, and electrophoretic mobility shift assays, we demonstrated that AGL16 was able to bind the CArG motifs in the promoters of the CYP707A3, AAO3, and SDD1 and regulate their transcription, leading to altered leaf stomatal density and ABA levels. Taking our findings together, AGL16 acts as a negative regulator of drought resistance by modulating leaf stomatal density and ABA accumulation.

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.

AtEDT1/HDG11 regulates stomatal density and water-use efficiency via ERECTA and E2Fa.

Improvement of crop drought resistance and water-use efficiency (WUE) has been a major endeavor in agriculture. Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a homeodomain-START transcription factor we previously identified from the enhanced drought tolerance1 mutant (edt1), has been demonstrated to improve drought tolerance and WUE significantly in multiple plant species when constitutively overexpressed. Here, we report the genetic evidence suggesting a genetic pathway, which consists of EDT1/HDG11, ERECTA, and E2Fa loci, and regulates WUE by modulating stomatal density. AtEDT1/HDG11 transcriptionally activates ERECTA by binding to homeodomain-binding (HD) cis-elements in the ERECTA promoter. ERECTA, in turn, depends on E2Fa to modulate the expression of cell cycle-related genes. This modulation affects the transition from mitosis to endocycle, leading to increased ploidy levels in leaf cells, and therefore increased cell size and decreased stomatal density. Our results suggest a possible EDT1/HDG11-ERECTA-E2Fa genetic pathway that reduces stomatal density by increasing cell size and provide a new avenue to improve WUE of crops.

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.].