Genetic and lipidomic analyses reveal the key role of lipid metabolism for cold tolerance in maize.

Lipid remodeling is crucial for cold tolerance in plants. However, the precise alternations of lipidomics during cold responses remain elusive, especially in maize (Zea mays L.). In addition, the key genes responsible for cold tolerance in maize lipid metabolism have not been identified. Here, we integrate lipidomic, transcriptomic, and genetic analysis to determine the profile of lipid remodeling caused by cold stress. We find that the homeostasis of cellular lipid metabolism is essential for maintaining cold tolerance of maize. Also, we detect 210 lipid species belonging to 13 major classes, covering phospholipids, glycerides, glycolipids, and free fatty acids. Various lipid metabolites undergo specific and selective alterations in response to cold stress, especially mono-/di-unsaturated lysophosphatidic acid, lysophosphatidylcholine, phosphatidylcholine, and phosphatidylinositol, as well as polyunsaturated phosphatidic acid, monogalactosyldiacylglycerol, diacylglycerol, and triacylglycerol. In addition, we identify a subset of key enzymes, including ketoacyl-acyl-carrier protein synthase II (KAS II), acyl-carrier protein 2 (ACP2), male sterility33 (Ms33), and stearoyl-acyl-carrier protein desaturase 2 (SAD2) involved in glycerolipid biosynthetic pathways are positive regulators of maize cold tolerance. These results reveal a comprehensive lipidomic profile during the cold response of maize and provide genetic resources for enhancing cold tolerance in crops.

Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.

How reciprocal regulation of carbon and nitrogen metabolism works is a long-standing question. In plants, glucose and nitrate are proposed to act as signaling molecules, regulating carbon and nitrogen metabolism via largely unknown mechanisms. Here, we show that the MYB-related transcription factor ARE4 coordinates glucose signaling and nitrogen utilization in rice. ARE4 is retained in the cytosol in complexing with the glucose sensor OsHXK7. Upon sensing a glucose signal, ARE4 is released, is translocated into the nucleus, and activates the expression of a subset of high-affinity nitrate transporter genes, thereby boosting nitrate uptake and accumulation. This regulatory scheme displays a diurnal pattern in response to circadian changes of soluble sugars. The are4 mutations compromise in nitrate utilization and plant growth, whereas overexpression of ARE4 increases grain size. We propose that the OsHXK7-ARE4 complex links glucose to the transcriptional regulation of nitrogen utilization, thereby coordinating carbon and nitrogen metabolism.

Nitric oxide-mediated S-nitrosylation of IAA17 protein in intrinsically disordered region represses auxin signaling.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. Auxin signaling is activated through the phytohormone-induced proteasomal degradation of the Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) family of transcriptional repressors. Notably, many auxin-modulated physiological processes are also regulated by nitric oxide (NO) that executes its biological effects predominantly through protein S-nitrosylation at specific cysteine residues. However, little is known about the molecular mechanisms in regulating the interactive NO and auxin networks. Here, we show that NO represses auxin signaling by inhibiting IAA17 protein degradation. NO induces the S-nitrosylation of Cys-70 located in the intrinsically disordered region of IAA17, which inhibits the TIR1-IAA17 interaction and consequently the proteasomal degradation of IAA17. The accumulation of a higher level of IAA17 attenuates auxin response. Moreover, an IAA17C70W nitrosomimetic mutation renders the accumulation of a higher level of the mutated protein, thereby causing partial resistance to auxin and defective lateral root development. Taken together, these results suggest that S-nitrosylation of IAA17 at Cys-70 inhibits its interaction with TIR1, thereby negatively regulating auxin signaling. This study provides unique molecular insights into the redox-based auxin signaling in regulating plant growth and development.

The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay.

Rapid production of H2O2 is a hallmark of plant responses to diverse pathogens and plays a crucial role in signalling downstream of various receptors that perceive immunogenic patterns. However, mechanisms by which plants sense H2O2 to regulate immunity remain poorly understood. We show that endogenous H2O2 generated upon immune activation is sensed by the thiol peroxidase PRXIIB via oxidation at Cys51, and this is essential for stomatal immunity against Pseudomonas syringae. We further show that in immune-stimulated cells, PRXIIB conjugates via Cys51 with the type 2C protein phosphatase ABA insensitive 2 (ABI2), subsequently transducing H2O2 signal to ABI2. This oxidation dramatically sensitizes H2O2-mediated inhibition of the ABI2 phosphatase activity in vitro and is required for stomatal immunity in plants. Together, our results illustrate a redox relay, with PRXIIB as a sensor for H2O2 and ABI2 as a target protein, that mediates reactive oxygen species signalling during plant immunity.

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker, a devastating disease threatening the Actinidia fruit industry. In a search for non-host resistance genes against Psa, we find that the nucleotide-binding leucine-rich repeat receptor (NLR) protein ZAR1 from both Arabidopsis and Nicotiana benthamiana (Nb) recognizes HopZ5 and triggers cell death. The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants, which are members of the ZRK pseudokinases and known components of the ZAR1 resistosome. Surprisingly, Arabidopsis ZAR1 and RPM1, another NLR known to recognize HopZ5, confer disease resistance to HopZ5 in a strain-specific manner. Thus, ZAR1, but not RPM1, is solely required for resistance to P. s. maculicola ES4326 (Psm) carrying hopZ5, whereas RPM1 is primarily required for resistance to P. s. tomato DC3000 (Pst) carrying hopZ5. Furthermore, the ZAR1-mediated resistance to Psm hopZ5 in Arabidopsis is insensitive to SOBER1, which encodes a deacetylase known to suppress the RPM1-mediated resistance to Pst hopZ5. In addition, hopZ5 enhances P. syringae virulence in the absence of ZAR1 or RPM1 and that SOBER1 abolishes such virulence function. Together the study suggests that ZAR1 may be used for improving Psa resistance in Actinidia and uncovers previously unknown complexity of effector-triggered immunity and effector-triggered virulence.

Nitric oxide negatively regulates gibberellin signaling to coordinate growth and salt tolerance in Arabidopsis.

In response to dynamically altered environments, plants must finely coordinate the balance between growth and stress responses for their survival. However, the underpinning regulatory mechanisms remain largely elusive. The phytohormone gibberellin promotes growth via a derepression mechanism by proteasomal degradation of the DELLA transcription repressors. Conversely, the stress-induced burst of nitric oxide (NO) enhances stress tolerance, largely relying on NO-mediated S-nitrosylation, a redox-based posttranslational modification. Here, we show that S-nitrosylation of Cys-374 in the Arabidopsis RGA protein, a key member of DELLAs, inhibits its interaction with the F-box protein SLY1, thereby preventing its proteasomal degradation under salinity condition. The accumulation of RGA consequently retards growth but enhances salt tolerance. We propose that NO negatively regulates gibberellin signaling via S-nitrosylation of RGA to coordinate the balance of growth and stress responses when challenged by adverse environments.

Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice.

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality. In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) cycle. How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RESPONSE1 (ABC1) gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome. Here, we show that ARE2 (for ABC1 REPRESSOR2) is a key regulator of nitrogen starvation responses in rice. The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency, suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT. ARE2 encodes a chloroplast-localized RelA/SpoT homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate (p)ppGpp, an alarmone regulating the stringent response in bacteria under nutritional stress conditions. The are2 mutants accumulate excessive amounts of (p)ppGpp, which correlate with lower levels of photosynthetic proteins and higher amino acid levels. Collectively, these observations suggest that the alarmone (p)ppGpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.

The Ghd7 transcription factor represses ARE1 expression to enhance nitrogen utilization and grain yield in rice.

The genetic improvement of nitrogen use efficiency (NUE) of crops is vital for grain productivity and sustainable agriculture. However, the regulatory mechanism of NUE remains largely elusive. Here, we report that the rice Grain number, plant height, and heading date7 (Ghd7) gene genetically acts upstream of ABC1 REPRESSOR1 (ARE1), a negative regulator of NUE, to positively regulate nitrogen utilization. As a transcriptional repressor, Ghd7 directly binds to two Evening Element-like motifs in the promoter and intron 1 of ARE1, likely in a cooperative manner, to repress its expression. Ghd7 and ARE1 display diurnal expression patterns in an inverse oscillation manner, mirroring a regulatory scheme based on these two loci. Analysis of a panel of 2656 rice varieties suggests that the elite alleles of Ghd7 and ARE1 have undergone diversifying selection during breeding. Moreover, the allelic distribution of Ghd7 and ARE1 is associated with the soil nitrogen deposition rate in East Asia and South Asia. Remarkably, the combination of the Ghd7 and ARE1 elite alleles substantially improves NUE and yield performance under nitrogen-limiting conditions. Collectively, these results define a Ghd7-ARE1-based regulatory mechanism of nitrogen utilization, providing useful targets for genetic improvement of rice NUE.

A route to de novo domestication of wild allotetraploid rice.

Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.

Transnitrosylation Mediated by the Non-canonical Catalase ROG1 Regulates Nitric Oxide Signaling in Plants.

The redox-based protein S-nitrosylation is a conserved mechanism modulating nitric oxide (NO) signaling and has been considered mainly as a non-enzymatic reaction. S-nitrosylation is regulated by the intracellular NO level that is tightly controlled by S-nitrosoglutathione reductase (GSNOR). However, the molecular mechanisms regulating S-nitrosylation selectivity remain elusive. Here, we characterize an Arabidopsis "repressor of" gsnor1 (rog1) mutation that specifically suppresses the gsnor1 mutant phenotype. ROG1, identical to the non-canonical catalase, CAT3, is a transnitrosylase that specifically modifies GSNOR1 at Cys-10. The transnitrosylase activity of ROG1 is regulated by a unique and highly conserved Cys-343 residue. A ROG1C343T mutant displays increased catalase but decreased transnitrosylase activities. Consistent with these results, the rog1 mutation compromises responses to NO under both normal and stress conditions. We propose that ROG1 functions as a transnitrosylase to regulate the NO-based redox signaling in plants.

An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence.

Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarm a pathogenic bacterium.

Two Plastid Fatty Acid Exporters Contribute to Seed Oil Accumulation in Arabidopsis.

Triacylglycerols (TAGs) are the major storage form of seed oil in oilseed plants. They are biosynthesized de novo in seed plastids and then transported into the endoplasmic reticulum. However, the transport mechanism for plastid fatty acids in developing seeds remains unknown. Here, we isolated two novel plastid fatty acid exporters (FATTYACID EXPORT 2 [FAX2] and FAX4, respectively) specifically abundant in seed embryos during the seed-filling stage in Arabidopsis (Arabidopsis thaliana). FAX2 and FAX4 were both localized to the chloroplast membrane. FAX2 and FAX4 loss-of-function mutations caused deficiencies in embryo and cotyledon development. Seeds of fax2fax4 double mutants exhibited significantly reduced TAG contents but elevated levels of plastid lipid contents compared with those of wild-type plants. By contrast, overexpression of FAX2 or FAX4 enhanced TAG deposition. Seed-feeding experiments showed that the two FAX proteins transported 14C-plastid fatty acids and 13C-oleic acids for TAG biosynthesis during the seed-filling stage. Together, our data demonstrate that FAX2 and FAX4 play critical roles in transporting plastid fatty acids for TAG biosynthesis during seed embryo development. These two transporters may have broad application for increasing oil yield in oilseed crops.

Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis.

Reactive oxygen species (ROS) play a crucial role in numerous biological processes in plants, including development, responses to environmental stimuli, and programmed cell death (PCD). Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase essential for de novo fatty acid biosynthesis in Arabidopsis thaliana, leads to the increased malate export from chloroplasts to mitochondria, and the subsequent accumulation of mitochondria-generated ROS and PCD. In this study, we report the identification and characterization of a mod1 suppressor, som592. SOM592 encodes mitochondrion-localized NAD+ transporter 2 (NDT2). We show that the mitochondrial NAD pool is elevated in the mod1 mutant. The som592 mutation fully suppressed mitochondrial NADH hyper-accumulation, ROS production, and PCD in the mod1 mutant, indicating a causal relationship between mitochondrial NAD accumulation and ROS/PCD phenotypes. We also show that in wild-type plants, the mitochondrial NAD+ uptake is involved in the regulation of ROS production in response to continuous photoperiod. Elevation of the alternative respiration pathway can suppress ROS accumulation and PCD in mod1, but leads to growth restriction. These findings uncover a regulatory mechanism for mitochondrial ROS production via NADH homeostasis in Arabidopsis thaliana that is likely important for growth regulation in response to altered photoperiod.

A new insight to explore the regulation between S-nitrosylation and N-glycosylation.

Nitric oxide (NO) is a signal molecule in plants and animals. Arabidopsis GSNO reductase1 (AtGSNOR1) catalyzes metabolism of S-nitrosoglutathione (GSNO) which is a major biologically active NO species. The GSNOR1 loss-of-function mutant gsnor1-3 overaccumulates GSNO with inherent high S-nitrosylation level and resistance to the oxidative stress inducer paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride). Here, we report the characterization of dgl1-3 as a genetic suppressor of gsnor1-3. DGL1 encodes a subunit of the oligosaccharyltransferse (OST) complex which catalyzes the formation of N-glycosidic bonds in N-glycosylation. The fact that dgl1-3 repressed the paraquat resistance of gsnor1-3 meanwhile gsnor1-3 rescued the embryo-lethal and post-embryonic development defect of dgl1-3 reminded us the possibility that S-nitrosylation and N-glycosylation crosstalk with each other through co-substrates. By enriching glycoproteins in gsnor1-3 and mass spectrometry analysis, TGG2 (thioglucoside glucohydrolase2) was identified as one of co-substrates with high degradation rate and elevated N-glycosylation level in gsnor1-3 ost3/6. The S-nitrosylation and N-glycosylation profiles were also modified in dgl1-3 and gsnor1-3. Thereby, we propose a linkage between S-nitrosylation and N-glycosylation through co-substrates.

Protein S-Nitrosylation in plants: Current progresses and challenges.

Nitric oxide (NO) is an important signaling molecule regulating diverse biological processes in all living organisms. A major physiological function of NO is executed via protein S-nitrosylation, a redox-based posttranslational modification by covalently adding a NO molecule to a reactive cysteine thiol of a target protein. S-nitrosylation is an evolutionarily conserved mechanism modulating multiple aspects of cellular signaling. During the past decade, significant progress has been made in functional characterization of S-nitrosylated proteins in plants. Emerging evidence indicates that protein S-nitrosylation is ubiquitously involved in the regulation of plant development and stress responses. Here we review current understanding on the regulatory mechanisms of protein S-nitrosylation in various biological processes in plants and highlight key challenges in this field.

S-Nitrosylation Targets GSNO Reductase for Selective Autophagy during Hypoxia Responses in Plants.

Nitric oxide (NO) regulates diverse cellular signaling through S-nitrosylation of specific Cys residues of target proteins. The intracellular level of S-nitrosoglutathione (GSNO), a major bioactive NO species, is regulated by GSNO reductase (GSNOR), a highly conserved master regulator of NO signaling. However, little is known about how the activity of GSNOR is regulated. Here, we show that S-nitrosylation induces selective autophagy of Arabidopsis GSNOR1 during hypoxia responses. S-nitrosylation of GSNOR1 at Cys-10 induces conformational changes, exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) accessible by autophagy machinery. Upon binding by ATG8, GSNOR1 is recruited into the autophagosome and degraded in an AIM-dependent manner. Physiologically, the S-nitrosylation-induced selective autophagy of GSNOR1 is relevant to hypoxia responses. Our discovery reveals a unique mechanism by which S-nitrosylation mediates selective autophagy of GSNOR1, thereby establishing a molecular link between NO signaling and autophagy.

Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.

Programmed cell death (PCD) is a fundamental biological process. Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase, leads to the accumulation of reactive oxygen species (ROS) and PCD, which can be suppressed by mitochondrial complex I mutations, indicating a signal from chloroplasts to mitochondria. However, this signal remains to be elucidated. In this study, through cloning and analyzing a series of mod1 suppressors, we reveal a comprehensive organelle communication pathway that regulates the generation of mitochondrial ROS and triggers PCD. We show that mutations in PLASTIDIAL NAD-DEPENDENT MALATE DEHYDROGENASE (plNAD-MDH), chloroplastic DICARBOXYLATE TRANSPORTER 1 (DiT1) and MITOCHONDRIAL MALATE DEHYDROGENASE 1 (mMDH1) can each rescue the ROS accumulation and PCD phenotypes in mod1, demonstrating a direct communication from chloroplasts to mitochondria via the malate shuttle. Further studies demonstrate that these elements play critical roles in the redox homeostasis and plant growth under different photoperiod conditions. Moreover, we reveal that the ROS level and PCD are significantly increased in malate-treated HeLa cells, which can be dramatically attenuated by knockdown of the human gene MDH2, an ortholog of Arabidopsis mMDH1. These results uncover a conserved malate-induced PCD pathway in plant and animal systems, revolutionizing our understanding of the communication between organelles.