The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

Abscisic acid-dependent PMT1 expression regulates salt tolerance by alleviating abscisic acid-mediated reactive oxygen species production in Arabidopsis.

Phosphocholine (PCho) is an intermediate metabolite of nonplastid plant membranes that is essential for salt tolerance. However, how PCho metabolism modulates response to salt stress remains unknown. Here, we characterize the role of phosphoethanolamine N-methyltransferase 1 (PMT1) in salt stress tolerance in Arabidopsis thaliana using a T-DNA insertional mutant, gene-editing alleles, and complemented lines. The pmt1 mutants showed a severe inhibition of root elongation when exposed to salt stress, but exogenous ChoCl or lecithin rescued this defect. pmt1 also displayed altered glycerolipid metabolism under salt stress, suggesting that glycerolipids contribute to salt tolerance. Moreover, pmt1 mutants exhibited altered reactive oxygen species (ROS) accumulation and distribution, reduced cell division activity, and disturbed auxin distribution in the primary root compared with wild-type seedlings. We show that PMT1 expression is induced by salt stress and relies on the abscisic acid (ABA) signaling pathway, as this induction was abolished in the aba2-1 and pyl112458 mutants. However, ABA aggravated the salt sensitivity of the pmt1 mutants by perturbing ROS distribution in the root tip. Taken together, we propose that PMT1 is an important phosphoethanolamine N-methyltransferase participating in root development of primary root elongation under salt stress conditions by balancing ROS production and distribution through ABA signaling.

Progress on methods for acquiring flanking genomic sequence.

Flanking genomic sequences refer to the DNA sequences flanking specific sites of known sequences in chromosome, which contain information such as candidate genes, transcriptional regulation, chromosome structure, and biosafety, and play an important role in genomics research. Flanking sequence acquisition technologies are mainly used in the cloning of regulatory sequences such as promoters and enhancers, identification of T-DNA or transposon insertion sites, chromosome walking, genome-wide gap filling, etc. It is an important means of structural genomics research and functional genomics research. It is applied in the identification of transgenic plants and animals and their safety management. With the development of molecular biology, many methods for obtaining flanking sequences have been established, including plasmid rescue, inverse PCR, ligation-mediated PCR, semi-random primer PCR, whole-genome resequencing etc. In this review, we summarize and compared different methods for acquiring flanking genomic sequence. The principles and research progress of each approach are discussed.

Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress.

BACKGROUND: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. RESULTS: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up- or down-regulated in roots of tomato following Al stress. CONCLUSION: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Ethylene promotes seed iron storage during Arabidopsis seed maturation via ERF95 transcription factor.

Because Iron (Fe) is an essential element, Fe storage in plant seeds is necessary for seedling establishment following germination. However, the mechanisms controlling seed Fe storage during seed development remain largely unknown. Here we reveal that an ERF95 transcription factor regulates Arabidopsis seed Fe accumulation. We show that expression of ERF95 increases during seed maturation, and that lack of ERF95 reduces seed Fe accumulation, consequently increasing sensitivity to Fe deficiency during seedling establishment. Conversely, overexpression of ERF95 has the opposite effects. We show that lack of ERF95 decreases abundance of FER1 messenger RNA in developing seed, which encodes Fe-sequestering ferritin. Accordingly, a fer1-1 loss-of-function mutation confers reduced seed Fe accumulation, and suppresses ERF95-promoted seed Fe accumulation. In addition, ERF95 binds to specific FER1 promoter GCC-boxes and transactivates FER1 expression. We show that ERF95 expression in maturing seed is dependent on EIN3, the master transcriptional regulator of ethylene signaling. While lack of EIN3 reduces seed Fe content, overexpression of ERF95 rescues Fe accumulation in the seed of ein3 loss-of-function mutant. Finally, we show that ethylene production increases during seed maturation. We conclude that ethylene promotes seed Fe accumulation during seed maturation via an EIN3-ERF95-FER1-dependent signaling pathway.

A Phosphate-Starvation Induced RING-Type E3 Ligase Maintains Phosphate Homeostasis Partially Through OsSPX2 in Rice.

Phosphate (Pi), as the main form of phosphorus that can be absorbed by plants, is one of the most limiting macro-nutrients for plants. However, the mechanism for maintaining Pi homeostasis in rice (Oryza sativa) is still not well understood. We identified a Pi-starvation-induced E3 ligase (OsPIE1) in rice. Using an in vitro self-ubiquitination assay, we determined the E3 ligase activities of OsPIE1. Using GUS staining and GFP detection, we analyzed tissue expression patterns of OsPIE1 and the subcellular localization of its encoded protein. The function of OsPIE1 in Pi homeostasis was analyzed using OsPIE1 overexpressors and ospie1 mutants. OsPIE1 was localized to the nucleus, and expressed in epidermis, exodermis and sclerenchyma layers of primary root. Under Pi-sufficient condition, overexpression of OsPIE1 upregulated the expression of OsPT2, OsPT3, OsPT10 and OsPAP21b, resulting in Pi accumulation and acid phosphatases (APases) induction in roots. OsSPX2 was strongly suppressed in OsPIE1 overexpressors. Further comparative transcriptome analysis, tissue expression patterns and genetic interaction analysis indicated that the enhancing of Pi accumulation and APase activities upon overexpression of OsPIE1 was (at least in part) caused by repression of OsSPX2. These results indicate that OsPIE1 plays an important role in maintaining Pi homeostasis in rice.

[Identifying T-DNA insertion site(s) of transgenic plants by whole-genome resequencing].

The availability of T-DNA insertion sites is very important for plant functional genomics research and the screening and identification of transgenic plants. However, the present protocols for identifying T-DNA insertion sites, like reverse PCR and semi-random primer PCR, are not only complex and time-consuming, but also inefficient. In this paper, a DNA pool of three transgenic plants was sequenced by whole-genome resequencing, and four T-DNA insertion sites were identified by blasting using transgenic T-DNA sequences. After PCR and Southern blot analysis, the T-DNA insertion sites of the three transgenic plants were successfully confirmed, and one of the transgenic plants showed two insertion sites. In conclusion, this study established a simple, reliable and efficient method for obtaining T-DNA insertion sites in transgenic plants.

[Relationship between Autophagy and Curcumin-induced Anticancer Effect].

Curcumin is a polyphenol extracted from turmeric rhizome and has multiple pharmacological roles. Recently,its anticancer properties have been recognized. Also,curcumin regulates autophagy in tumor cells via signaling pathways including AMP-activated protein kinase,mammalian target of rapamycin,transcription factor EB,Beclin-1,B-cell lymphoma 2,and endoplasmic reticulum stress. Considering the complicated crosstalk between autophagy and apoptosis,in this article we summaize the mechanism of curcumin-induced autophagy and its effect on apoptosis,with an attempt to provide insights on tumor therapy.

OsCLT1, a CRT-like transporter 1, is required for glutathione homeostasis and arsenic tolerance in rice.

Arsenic (As) contamination in a paddy environment can cause phytotoxicity and elevated As accumulation in rice (Oryza sativa). The mechanism of As detoxification in rice is still poorly understood. We isolated an arsenate (As(V))-sensitive mutant of rice. Genomic resequencing and complementation identified OsCLT1, encoding a CRT-like transporter, as the causal gene for the mutant phenotype. OsCLT1 is localized to the envelope membrane of plastids. The glutathione and γ-glutamylcysteine contents in roots of Osclt1 and RNA interference lines were decreased markedly compared with the wild-type (WT). The concentrations of phytochelatin PC2 in Osclt1 roots were only 32% and 12% of that in WT after As(V) and As(III) treatments, respectively. OsCLT1 mutation resulted in lower As accumulation in roots but higher As accumulation in shoots when exposed to As(V). Under As(III) treatment, Osclt1 accumulated a lower As concentration in roots but similar As concentration in shoots to WT. Further analysis showed that the reduction of As(V) to As(III) was decreased in Osclt1. Osclt1 was also hypersensitive to cadmium (Cd). These results indicate that OsCLT1 plays an important role in glutathione homeostasis, probably by mediating the export of γ-glutamylcysteine and glutathione from plastids to the cytoplasm, which in turn affects As and Cd detoxification in rice.

A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate inositol hexaphosphate accumulation.

Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.

The Arabidopsis receptor-like kinase WAKL4 limits cadmium uptake via phosphorylation and degradation of NRAMP1 transporter.

Cadmium (Cd) is a detrimental heavy metal propagated from soil to the food chain via plants, posing a great risk to human health upon consumption. Despite the understanding of Cd tolerance mechanisms in plants, whether and how plants actively respond to Cd and in turn restrict its uptake and accumulation remain elusive. Here, we identify a cell wall-associated receptor-like kinase 4 (WAKL4) involved in specific tolerance to Cd stress. We show that Cd rapidly and exclusively induces WAKL4 accumulation by promoting WAKL4 transcription and blocking its vacuole-dependent proteolysis in roots. The accumulated WAKL4 next interacts with and phosphorylates the Cd transporter NRAMP1 at Tyr488, leading to the enhanced ubiquitination and vacuole-dependent degradation of NRAMP1, and consequently reducing Cd uptake. Our findings therefore uncover a mechanism conferred by the WAKL4-NRAMP1 module that enables plants to actively respond to Cd and limit its uptake, informing the future molecular breeding of low Cd accumulated crops or vegetables.

An LRH-RSL4 feedback regulatory loop controls the determinate growth of root hairs in Arabidopsis.

Root hairs are tubular-shaped outgrowths of epidermal cells essential for plants acquiring water and nutrients from the soil. Despite their importance, the growth of root hairs is finite. How this determinate growth is precisely regulated remains largely unknown. Here we identify LONG ROOT HAIR (LRH), a GYF domain-containing protein, as a unique repressor of root hair growth. We show that LRH inhibits the association of eukaryotic translation initiation factor 4Es (eIF4Es) with the mRNA of ROOT HAIR DEFECTIVE6-LIKE4 (RSL4) that encodes the master regulator of root hair growth, repressing RSL4 translation and thus root hair elongation. RSL4 in turn directly transactivates LRH expression to maintain a proper LRH gradient in the trichoblasts. Our findings reveal a previously uncharacterized LRH-RSL4 feedback regulatory loop that limits root hair growth, shedding new light on the mechanism underlying the determinate growth of root hairs.

The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

Diselenide-Based Dual-Responsive Prodrug as Pyroptosis Inducer Potentiates Cancer Immunotherapy.

Pyroptosis is demonstrated to trigger antitumor immunity and represents a promising new strategy to potentiate cancer immunotherapy. The number of potent pyroptosis inducers, however, is limited and without tumor-targeting capability, which inevitably causes damage in normal tissues. Herein, a small molecular prodrug of paclitaxel-oxaliplatin is rationally synthesized, which can be covalently self-assembled with diselenide-containing cross-linking (Dse11), producing a diselenide nanoprodrug (DSe@POC) to induce pyroptosis for the first time. The diselenide bonds within DSe@POC can be split by high glutathione in the tumor microenvironment (TME) and reactive oxygen species induced by photodynamic therapy, thus possessing excellent TME on-target effects. Additionally, DSe@POC is able to elicit intense pyroptosis to remodel the immunostimulated TME and trigger a robust immune response. Furthermore, combined αPD-1 therapy effectively inhibits the growth of remote tumors through the abscopal effect, amplifies a long-term immune memory response to reject rechallenged tumors, and prolongs survival. Collectively, DSe@POC, as the first TME dual-responsive diselenide-based pyroptosis inducer, will open up an attractive approach for cancer immunotherapy.

The Tomato Transcription Factor SlNAC063 Is Required for Aluminum Tolerance by Regulating SlAAE3-1 Expression.

Aluminum (Al) toxicity constitutes one of the major limiting factors of plant growth and development on acid soils, which comprises approximately 50% of potentially arable lands worldwide. When suffering Al toxicity, plants reprogram the transcription of genes, which activates physiological and metabolic pathways to deal with the toxicity. Here, we report the role of a NAM, ATAF1, 2 and CUC2 (NAC) transcription factor (TF) in tomato Al tolerance. Among 53 NAC TFs in tomatoes, SlNAC063 was most abundantly expressed in root apex and significantly induced by Al stress. Furthermore, the expression of SlNAC063 was not induced by other metals. Meanwhile, the SlNAC063 protein was localized at the nucleus and has transcriptional activation potentials in yeast. By constructing CRISPR/Cas9 knockout mutants, we found that slnac063 mutants displayed increased sensitivity to Al compared to wild-type plants. However, the mutants accumulated even less Al than wild-type (WT) plants, suggesting that internal tolerance mechanisms but not external exclusion mechanisms are implicated in SlNAC063-mediated Al tolerance in tomatoes. Further comparative RNA-sequencing analysis revealed that only 45 Al-responsive genes were positively regulated by SlNAC063, although the expression of thousands of genes (1,557 upregulated and 636 downregulated) was found to be affected in slnac063 mutants in the absence of Al stress. The kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that SlNAC063-mediated Al-responsive genes were enriched in "phenylpropanoid metabolism," "fatty acid metabolism," and "dicarboxylate metabolism," indicating that SlNAC063 regulates metabolisms in response to Al stress. Quantitative real-time (RT)-PCR analysis showed that the expression of SlAAE3-1 was repressed by SlNAC063 in the absence of Al. However, the expression of SlAAE3-1 was dependent on SlNAC063 in the presence of Al stress. Taken together, our results demonstrate that a NAC TF SlNAC063 is involved in tomato Al tolerance by regulating the expression of genes involved in metabolism, and SlNAC063 is required for Al-induced expression of SlAAE3-1.

Restriction of iron loading into developing seeds by a YABBY transcription factor safeguards successful reproduction in Arabidopsis.

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression.

A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis.

Phosphorus (P) is an indispensable macronutrient required for plant growth and development. Natural phosphate (Pi) reserves are finite, and a better understanding of Pi utilization by crops is therefore vital for worldwide food security. Ammonium has long been known to enhance Pi acquisition efficiency in agriculture; however, the molecular mechanisms coordinating Pi nutrition and ammonium remains unclear. Here, we reveal that ammonium is a novel initiator that stimulates the accumulation of a key regulatory protein, STOP1, in the nuclei of Arabidopsis root cells under Pi deficiency. We show that Pi deficiency promotes ammonium uptake mediated by AMT1 transporters and causes rapid acidification of the root surface. Rhizosphere acidification-triggered STOP1 accumulation activates the excretion of organic acids, which help to solubilize Pi from insoluble iron or calcium phosphates. Ammonium uptake by AMT1 transporters is downregulated by a CIPK23 protein kinase whose expression is directly modulated by STOP1 when ammonium reaches toxic levels. Taken together, we have identified a STOP1-centered regulatory network that links external ammonium with efficient Pi acquisition from insoluble phosphate sources. These findings provide a framework for developing possible strategies to improve crop production by enhancing the utilization of non-bioavailable nutrients in soil.

[Construction of a novel vector harboring green fluorescence protein gene (gfp) and high expression of gfp in transformed roots of Petunia hybrida].

A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida. The high activity of green fluo-rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5' and 3' of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloning sites at 5' end and one-sites of EcoRand Bsmsites at 3' end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transforma-tion.