Overexpression Lines Research Articles

OsLdh7, a rice lactate dehydrogenase, confers stress resilience in rice under cadmium stress through NAD+/NADH regulation

Lactate dehydrogenase (Ldh, EC 1.1.1.27), an oxidoreductase enzyme catalyses the interconversion of pyruvate to L-lactate and vice-versa with concomitant oxidation and reduction of NADH and NAD+. The enzyme functions as a ROS sensor and mitigates stress response by maintaining NAD+/NADH homeostasis. In this study, we delineated the role of the Ldh enzyme in imparting cadmium stress tolerance in rice. Previously, we identified a putatively active Ldh in rice (OsLdh7) through insilico modelling. Biochemical characterization of the OsLdh7 enzyme revealed it to be optimally active at pH 6.6 in the forward direction and pH 9 in the reverse direction. Overexpression of OsLdh7 in rice cv. IR64, increased tolerance of the transgenic lines to cadmium stress compared to the wild type (WT) at both seedling and reproductive stages. The transgenic lines showed increased enzyme activity in the reverse direction under cadmium stress, attributed to elevated cytosolic pH resulting from increased calcium concentration. This increased NADH content is highly essential for functioning of the ROS scavenging enzymes, RbohD and MPK6. qPCR analysis revealed that the overexpression lines had increased transcript abundance of these genes indicating an effective ROS scavenging mechanism. Additionally, the overexpression lines showed an efficient cadmium sequestration mechanism compared to the WT by increasing the transcript levels of the vacuolar transporters of cadmium as well as total phytochelatin content. Thus, our findings indicated OsLdh7 imparts cadmium stress tolerance in rice through a two-pronged approach by mitigating ROS and sequestering cadmium ions, highlighting its potential for crop improvement programs.

CircGNAO1 suppresses hepatocellular carcinoma progression and metastasis via sponging miR-182-5p and regulating FOXO1 expression

BackgroundHepatocellular carcinoma (HCC) is an aggressive malignant tumor with poor prognosis. Using high-throughput sequencing, we identified a novel circRNA, circGNAO1, which is downregulated in HCC tissues compared to adjacent tissues. However, the potential functions and mechanisms of circGNAO1 in HCC metastasis remain unclear. MethodsqRT-PCR was used to detect the expression of circGNAO1, miR-182-5p, and FOXO1 in HCC cells and tissues. Bioinformatics analysis, RNA pull-down assyas, and dual-luciferase reporter assays were employed to verify the interaction between circGNAO1 and miR-182-5p. Functional experiments were conducted using circGNAO1 overexpression and knockdown cell lines, including Transwell, wound healing, and EdU assays. Liver metastasis models and subcutaneous xenograft mouse models were established to analyze the effect of circGNAO1 on HCC metastasis and growth in vivo. ResultsHigh-throughput sequencing and qRT–PCR results showed that the expression of circGNAO1 dramatically decreased in HCC tissues. Functionally, in vivo and in vitro experiments verified that overexpression of circGNAO1 inhibited the proliferation, migration, invasion and EMT of HCC cells, while knockdown of circGNAO1 promoted these behaviors. Mechanistically, we have demonstrated that circGNAO1 functions as a sponge for miR-182-5p to regulate FOXO1 expression, thereby activating the TGF-β/Smad3 signaling pathway and EMT process. ConclusionscircGNAO1 suppresses the progression and metastasis of HCC through the miR-182-5p/FOXO1 axis, and circGNAO1 may be an efficient therapeutic target in HCC.

GmIRT1.1 from soybean (Glycine max L.) is involved in transporting Fe, Mn and Cd

Soybean is one of the most important crops for producing high quality oil and protein. Mineral nutrient deficiencies are frequently observed in soybeans. However, there are few studies to understand the absorption process of mineral nutrients in soybeans. Here, we investigated the functions of soybean (Glycine max L.) IRT1.1 (IRON-REGULATED TRANSPORTER 1.1) in the transportation of mineral elements. Heterologous expression of GmIRT1.1 in yeast mutants revealed that GmIRT1.1 compensated for the growth defects of Δfet3fet4 and Δsmf1 mutants under iron (Fe) and manganese (Mn) deficiency conditions, respectively, and enhanced the sensitivity of the Δycf1 mutant to cadmium (Cd) toxicity. Expression analysis revealed that GmIRT1.1 was only significantly induced by Fe deficiency and was primarily expressed in roots. Furthermore, the GmIRT1.1 overexpression lines enhanced Arabidopsis tolerance to Fe deficiency, leading to increased accumulation of Fe in the roots and shoots. Additionally, the transgenic lines increased the sensitivity to Mn and Cd toxicity. Subcellular localization analysis revealed that GmIRT1.1 was localized on the plasma membrane. Moreover, the results obtained from the soybean hairy roots system indicated that the localization of GmIRT1.1 was dependent on the regulation of Fe homeostasis in plant. Consequently, these results suggested that GmIRT1.1 was responsible for the transportation of Fe, Mn and Cd.

Overexpression of EaALDH7, an aldehyde dehydrogenase gene from Erianthus arundinaceus enhances salinity tolerance in transgenic sugarcane (Saccharum spp. Hybrid)

Aldehyde Dehydrogenases (ALDH), a group of enzymes, are associated with the detoxification of aldehydes, produced in plants during abiotic stress conditions. Salinity remains a pivotal abiotic challenge that poses a significant threat to cultivation and yield of sugarcane. In this study, an Aldehyde dehydrogenase gene (EaALDH7) from Erianthus arundinaceus was overexpressed in the commercial sugarcane hybrid cultivar Co 86032. The transgenic lines were evaluated at different NaCl concentrations ranging from 0 mM to 200 mM for various morpho-physiological and biochemical parameters. The control plants, subjected to salinity stress condition, exhibited morphological changes in protoxylem, metaxylem, pericycle and pith whereas the transgenic events were on par with plants under regular irrigation. The overexpressing (OE) lines showed less cell membrane injury and improved photosynthetic rate, transpiration rate, and stomatal conductance than the untransformed control plants under stress conditions. Elevated proline content, higher activity of enzymatic antioxidants such as sodium dismutase (SOD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) and low level of malondialdehyde MDA and hydrogen peroxide (H2O2) in the transgenic lines. The analysis of EaALDH7 expression revealed a significant upregulation in the transgenic lines compared to that of the untransformed control during salt stress conditions. The current study highlights the potentials of EaALDH7 gene in producing salinity-tolerant sugarcane cultivars.

Abstract Or125: Numb Family Proteins regulate left ventricular compaction by modulating autophagic process

Background: Left ventricular noncompaction (LVNC) is a type of cardiomyopathy, which patients manifest clinical features of heart failure, arrhythmias, thrombotic events, and sudden death. We previously found that the ablation of Numb(Nb) , and its homologue Numbl(Nl) at early stage, causes LVNC defects resulting in embryonic lethality. In this study, we found that mice with Nb and Nl deletion at later stage survive to adulthood and develop toLVNC cardiomyopathy. Method: We applied aMHC-Cre to delete and generate Nb and Nl double knockout (ADKO); examine the structure and functions of the ADKO hearts via echocardiography (ECHO); determine the heart arrhythmia via telemetry; determine the Nb subcellular localization via a Nb overexpression line (R26-Flag:mCherry:Numb); and determine the mechanistic function of NFPs in autophagy via autophagic reporter, electron microscopy, and molecular and biochemical assays. Results: The cTnT-Cre mediated Nb and Nl deletion causes LVNC and embryonic lethality, while the ADKO can survive to adulthood and died between eight- and twelve-month-old. The ADKO hearts display LVNC by two months of age, a reduced systolic function by four months old, and left ventricular remodeling and dilatation starting at six months old. ADKO hearts manifest major clinical features of LVNC, including arrhythmias and thrombosis. ADKO hearts exhibit significant accumulation of damaged mitochondria, autophagosomes and autophagolysosomes compared to controls. Biochemical analyses reveal elevated levels of p62 and a higher ratio of LC3II/LC3I in ADKO hearts. NFP null cells and ADKO hearts exhibit abnormal autophagic flux, characterized by arrested at autophagolysosome, increased expression of Lamp1 and Lamp2, but reduced levels of cathepsin D. Mass spectrometry analyses reveal Numb's interaction with proteins involved in endocytosis and autophagy. Consistently, Numb localizes to autophagosomes and autophagolysosomes. Conclusion: NFPs regulate left ventricular compaction at both embryonic and postnatal stages, suggesting that LVNC can be both congenital and acquired. As LVNC mice progress, they develop dilated cardiomyopathy (DCM) at later stage, suggesting that LVNC can occur independently at early stage, and is associated with DCM at later stage. Deletion of NFPs in cardiomyocytes disrupts endocytosis and autophagic flux. Our studies suggest that NFPs mediated endocytosis and autophagy are essential for left ventricular compaction.

An ABC transporter-mediated transport and metabolism of the pesticide bentazone in rice (Oryza sativa L.)

IntroductionBentazon (BNTZ) is a selective contact herbicide widely used to control field weeds for crop production. Excessive use of BNTZ leads to its accumulation in soils and crops, becoming an environmental contaminant. Therefore, investigation of the mechanisms for BNTZ detoxification and degradation in crops is fundamentally important to reduce crop contamination and ensure food safety. ObjectivesThis study aims to elucidate the mechanism of detoxification and degradation pathways of the BNTZ complex in rice by creating transgenic lines expressing a rice ATP-binding cassette (OsABC) transporter gene through genetic engineering techniques combined with chemical analytical techniques and metabolomics approaches. MethodsWe established the rice transgenic lines overexpressing (OE) a rice OsABC transporter and its knockout lines by CRISPR-Cas9 to characterize the gene function and measured the accumulation of BNTZ residues in rice. The metabolites of BNTZ were characterized by LC/Q-TOF-HRMS/MS (Liquid chromatography/time of flight-high resolution mass spectrometry). ResultsOverexpression of OsABC significantly conferred rice resistance to BNTZ toxicity by increasing plant elongation, dry weight, and chlorophyll content, and significantly reducing cell membrane damage and BNTZ accumulation in rice tissues. Six different metabolites and ten conjugates were well defined in chemical structures. The reduced BNTZ levels and degradation products in the grains of the OE lines supported the robust activity of the OsABC gene function.Using UPLC-Q-TOF/MS, we further identified accumulated basic metabolites of various carbohydrates, amino acids, hormones, and flavonoids, and found that these metabolites involved in BNTZ degradation were increased more in OE lines than in wild-type (WT) rice. ConclusionsOur work demonstrates that the OsABC transporter plays a critical role in regulating the mobility and degradative metabolism of BNTZ in rice, thus revealing a regulatory mechanism underlying rice resistance to BNTZ toxicity and adaptation to the environmental stress.

Leaf starch degradation by β-amylase ZmBAM8 influences drought tolerance in maize

As a typical C4 plant and important crop worldwide, maize is susceptible to drought. In maize, transitory starch (TS) turnover occurs in the vascular bundle sheath of leaves, differing from that in Arabidopsis (a C3 plant). This process, particularly its role in drought tolerance and the key starch-hydrolyzing enzymes involved, is not fully understood. We discovered that the expression of the β-amylase (BAM) gene ZmBAM8 is highly upregulated in the drought-tolerant inbred line Chang7-2t. Inspired by this finding, we systematically investigated TS degradation in maize lines, including Chang7-2t, Chang7-2, B104, and ZmBAM8 overexpression (OE) and knockout (KO) lines. We found that ZmBAM8 was significantly induced in the vascular bundle sheath by drought, osmotic stress, and abscisic acid. The stress-induced gene expression and chloroplast localization of ZmBAM8 align with the tissue and subcellular sites where TS turnover occurs. The recombinant ZmBAM8 was capable of effectively hydrolyzing leaf starch. Under drought conditions, the leaf starch in ZmBAM8-OE plants substantially decreased under light, while that in ZmBAM8-KO plants did not decrease. Compared with ZmBAM8-KO plants, ZmBAM8-OE plants exhibited increased drought tolerance. Our study provides insights into the significance of leaf starch degradation in C4 crops and contributes to the development of drought-resistant maize.

Orphan gene BR2 positively regulates bolting resistance through the vernalization pathway in Chinese cabbage.

Orphan genes (OGs) are unique to the specific species or lineage, and whose homologous sequences cannot be found in other species or lineages. Furthermore, these genes lack recognizable domains or functional motifs, which make their characterization difficult. Here, we identified a Brassica rapa OG named BOLTING RESISTANCE 2 (BR2) that could positively modulate bolting resistance. The expression of BR2 was developmentally regulated and the BR2 protein was localized to the cell membrane. BR2 overexpression not only markedly delayed flowering time in Arabidopsis transgenic plants, but substantially affected the development of leaves and flower organs. Flowering repressor AtFLC gene was significantly up-regulated transcribed in Arabidopsis BR2 overexpression lines, while AtFT and AtSOC1 expression was decreased. In addition, the BR2 expression was enhanced in bolting-resistant type Chinese cabbage and was reduced in non-resistant type. Moreover, chilling stress inhibited the BR2 expression levels. Overexpression of BR2 also delayed flowering time in Chinese cabbage. In vernalized Chinese cabbage BR2 overexpression plants, BrVIN3.b and BrFRI were significantly down-regulated, while BrFLC5 was substantially up-regulated. Key floral factors, including three BrSOC1s, two BrLFYs, and four BrFTs were down-regulated. The expression changes of these key genes were consistent with the delayed flowering phenotype of Chinese cabbage BR2 overexpressing plants. Thus, we predicted that BR2 may predominantly function via the vernalization pathway. Our findings propose that the OG BR2 acts as a novel modulator of flowering time in Chinese cabbage, which provides a new insight on the breeding of varieties that are resistant to bolting.

Balancing roles between phosphatidylinositols and sphingolipids in regulating immunity and ER stress responses in pi4kβ1,2.

Plant immune regulation is complex. In addition to proteins, lipid molecules play critical roles in modulating immune responses. The mutant pi4kβ1,2 is mutated in two phosphatidylinositol 4-kinases PI4Kβ1 and β2 involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P). The mutant displays autoimmunity, short roots, aberrant root hairs, and a heightened sensitivity to ER stress. In a forward genetic screen designed to dissect pi4kβ1,2 autoimmunity, we found that Orosomucoid-like 1 (ORM1) is required for the phenotypes of pi4kβ1,2, including short root and ER stress sensitivity. The orm1 mutations lead to increased long-chain base and ceramide levels in the suppressors. We also found that the basic region/leucine Zipper motif (bZIP) 28 and 60 transcription factors, central regulators of ER stress response, are required for its autoimmunity and root defect. In comparison, the defense-related phytohormones salicylic acid (SA) and N-hydroxypipecolic acid (NHP) are required for its autoimmunity but plays a minor role in its root phenotypes. Further, we found that wild-type plants overexpressing ORM1 are autoimmune, displaying short roots and increased ceramide levels. The autoimmunity of the ORM1 overexpression lines is dependent on SA, NHP, and bZIP60. As ORM1 is a known negative regulator of sphingolipid biosynthesis, our study uncovers a balancing role between PIs and sphingolipids in regulating immunity and ER stress responses in pi4kβ1,2.

The U1 small nuclear RNA enhances drought tolerance in Arabidopsis.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism that improves plant tolerance to drought stress by modulating gene expression and generating proteome diversity. The interaction between the 5' end of U1 small nuclear RNA (U1 snRNA) and the conserved 5' splice site of precursor messenger RNA (pre-mRNA) is pivotal for U1 snRNP involvement in AS. However, the roles of U1 snRNA in drought stress responses remain unclear. This study provides a comprehensive analysis of AtU1 snRNA in Arabidopsis (Arabidopsis thaliana), revealing its high conservation at the 5' end and a distinctive four-leaf clover structure. AtU1 snRNA is localized in the nucleus and expressed in various tissues, with prominent expression in young floral buds, flowers, and siliques. Overexpression of AtU1 snRNA confers enhanced abiotic stress tolerance, as evidenced in seedlings by longer seedling primary root length, increased fresh weight, and a higher greening rate compared to the wild type. Mature AtU1 snRNA overexpressor plants exhibit higher survival rates and lower water loss rates under drought stress, accompanied by a significant decrease in H2O2 and increase in proline. This study also provides evidence of altered expression levels of drought-related genes in AtU1 snRNA overexpressor or genome-edited lines, reinforcing the crucial role of AtU1 snRNA in drought stress responses. Furthermore, the overexpression of AtU1 snRNA influences the splicing of downstream target genes, with a notable impact on SPEECHLESS (SPCH), a gene associated with stomatal development, potentially explaining the observed decrease in stomatal aperture and density. These findings elucidate the critical role of U1 snRNA as an AS regulator in enhancing drought stress tolerance in plants, contributing to a deeper understanding of the AS pathway in drought tolerance and increasing awareness of the molecular network governing drought tolerance in plants.

Plant cadmium resistance 2.7 from Brassica napus (BnPCR2.7) improves copper and cadmium tolerance

Copper (Cu) and cadmium (Cd) are highly phytotoxic heavy metals that are widespread contaminants in soil. Plants are efficient at taking up heavy metals, which adversely impacts human health. Therefore, it is important to decrease the accumulation of Cu/Cd in plants to reduce human exposure from the food web. Here, we determined the function of a rapeseed (Brassica napus) Cu/Cd transporter, plant cadmium resistance protein 2.7 (BnPCR2.7), in enhancing Cu/Cd tolerance in seedlings and decreasing the accumulation of Cu/Cd in seeds. A subcellular localization analysis revealed that BnPCR2.7 is localized at the plasma membrane (PM). CRISPR/Cas9-mediated BnPCR2.7 knockout lines and knockdown lines had increased sensitivity to high Cu/Cd than wild-type (WT) plants. In contrast, overexpression of BnPCR2.7 enhanced the adaptation to high Cu/Cd than in WT. Additionally, the Cu/Cd content in the roots of overexpression lines was significantly lower than those of the WT, while the contents in the stems increased. A non-invasive micro-test technology (NMT) assay showed that overexpression plants promoted the efflux of Cu and Cd from the roots. A field trial of rapeseed grown in soil contaminated with Cu or Cd showed that overexpression plants grew and developed better than the WT with higher yields and less Cu/Cd that accumulated in the seeds, while knockout and knockdown lines were contrary to these results. Additionally, when grown in soils contaminated by both Cu and Cd, the content of these heavy metals decreased by 12–20 % and 20–30 %, respectively, in seeds of overexpression lines. Collectively, BnPCR2.7 may promotes resistance to Cu/Cd by efflux pathways. Significantly, it is a candidate genetic resource to increase the resistance to heavy metals and reduce the accumulation of Cu/Cd in rapeseed.

Amino acid permease OsAAP12 negatively regulates rice tillers and grain yield by transporting specific amino acids to affect nitrogen and cytokinin pathways

Amino acids are necessary nutrients for the growth of Oryza sativa (rice), which can be mediated by amino acid transporter; however, our understanding of these transporters is still limited. This study found that the expression levels of amino acid permease gene OsAAP12 differed between indica and japonica rice. Altered expression of OsAAP12 negatively regulated tillering and yield in transgenic rice lines. Subcellular localization revealed that OsAAP12 was primarily localized to the plasma membrane. Moreover, it was indicated that OsAAP12 transported polar neutral amino acids asparagine (Asn), threonine (Thr), and serine (Ser) through experiments involving yeast heterologous complementation, fluorescence amino acid uptake, and amino acid content determination. Additionally, exogenous application of amino acids Asn, Thr, and Ser suppressed axillary buds outgrowth in OsAAP12 overexpression lines compared with wild-type ZH11. Conversely, the opposite trend was observed in CRISPR mutant lines. RNA-seq analysis showed that the expression patterns of genes involved in the nitrogen and cytokinin pathways were generally altered in OsAAP12 modified lines. Hormone assays indicated that OsAAP12 mutant lines accumulated cytokinins in the basal part of rice, whereas overexpression lines had the opposite effect. In summary, CRISPR mutant of OsAAP12 boosted rice tillering and grain yield by coordinating the content of amino acids and cytokinins, which has potential application value in high-yield rice breeding.

GmAMT2.1/2.2-dependent ammonium nitrogen and metabolites shape rhizosphere microbiome assembly to mitigate cadmium toxicity

Cadmium (Cd), a heavy metal, is negatively associated with plant growth. AMT (ammonium transporter) genes can confer Cd resistance and enhance nitrogen (N) uptake in soybeans. The potential of AMT genes to alleviate Cd toxicity by modulating rhizosphere microbiota remains unkonwn. Here, the rhizosphere microbial taxonomic and metabolic differences in three genotypes, i.e., double knockout and overexpression lines and wild type, were identified. The results showed that GmAMT2.1/2.2 genes could induce soybean to recruit beneficial microorganisms, such as Tumebacillus, Alicyclobacillus, and Penicillium, by altering metabolites. The bacterial, fungal, and cross-kingdom synthetic microbial communities (SynComs) formed by these microorganisms can help soybean resist Cd toxicity. The mechanisms by which SynComs help soybeans resist Cd stress include reducing Cd content, increasing ammonium (NH4+-N) uptake and regulating specific functional genes in soybeans. Overall, this study provides valuable insights for the developing microbial formulations that enhance Cd resistance in sustainable agriculture.

StSnRK1.1 protein kinase positively regulates tuber dormancy release of potato

SnRK1 is a plant-specific serine/threonine protein kinase, that plays a vital role in maintaining the energy balance, nutrient distribution, growth, and development of organisms for survival. The period of tuber dormancy seriously affects potato production. To elucidate the effect of the StSnRK1.1 gene on dormancy release, the gene was cloned from the potato cultivar 'Desiree', and the overexpression and interfering expression lines were obtained through genetic transformation. StSnRK1.1 promoted early dormancy release and sprouting of potato tubers in the overexpression line, while it delayed tuber sprouting in the StSnRK1.1-interfering expression line. In addition, it was discovered that abscisic acid (ABA) in the StSnRK1.1 overexpression line was significantly reduced compared with the wild type plants, while the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased. Conversely, the opposite result was found in the StSnRK1.1 gene interference expression line, which indicated that the dormancy release was accelerated, the reactive oxygen species metabolism efficiency was high, and the reactive oxygen species could be quickly removed and the accumulation of reactive oxygen species could be avoided during the dormancy release process. The positive regulatory genes StSnRK2.2 and StABI5 of ABA signal transduction were lower than those of wild type in the overexpression line and higher than that of the wild type in the interfering expression line, while the negative regulatory genes StPP2CA1 and StPP2CA3 of ABA signal transduction were higher than those of the wild type in the overexpression line and lower than that of the wild type in the interfering expression line. In summary, StSnRK1.1 affects ABA signaling by mediating the expression of StSnRK2.2, StABI5, StPP2CA1 and StPP2CA3, which provides a theoretical basis for the molecular mechanism of StSnRK1.1 to positively regulate the release of potato tuber dormancy. Through yeast two-hybrid (YH2) screening, two interacting proteins, StCAT3 and StFBA1, were identified. The interaction between StCAT3, StFBA1 and StSnRK1.1 was confirmed through bimolecular fluorescence complementation. These results provide novel information for further study on the molecular mechanism of StSnRK1.1 regulating dormancy and germination of potato tubers.

Xyloglucan endotransglucosylase-hydrolase 22 positively regulates response to cold stress in upland cotton (Gossypium hirsutum L.)

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall modifying enzymes that function in response to multiple abiotic stresses by promoting cell wall reinforcement in plants. However, it is elusive whether GhXTH22 plays a role in cold stress tolerance in upland cotton (Gossypium hirsutum L.). In this study, we found that Arabidopsis (Arabidopsis thaliana (L.) Heynh) transgenic lines overexpressing GhXTH22 exhibit clearly increased cold tolerance compared with wild type (WT). Moreover, cold stress results in markable enhancements in the activities of catalase (CAT) and peroxidase (POD), cell wall rigidity, and the expression of cold responsive genes, as well as a notable reduction in malondialdehyde (MDA) levels in the overexpression (OE) lines compared with those in WT plants. Consistently, silencing GhXTH22 by virus-induced gene silencing (VIGS) method causes markedly decreases in cold stress tolerance in upland cotton. Additionally, by electrophoretic mobility shift assay (EMSA) and dual-luciferase assay, it was confirmed that GhMYB44 activate the expression of GhXTH22 by binding to AACNG motif on the promoter, and silencing the expression of GhMYB44 can decrease the cold resistance of cotton. In summary, this study systematically confirmed the cold resistance function of GhXTH22 and discovered the upstream regulating gene of GhXTH22, providing gene resources for cold resistance and enriching the knowledge on the mechanism of cold resistance.

Phytochrome interacting factor ZmPIF6 simultaneously enhances chilling tolerance and grain size in rice

Chilling is a prevalent type of abiotic stress that adversely affects agricultural productivity worldwide. Phytochrome interacting factors (PIFs) are a group of transcription factor that are crucial for plant abiotic stress response. Our research reveals that the maize PIF family gene ZmPIF6 is responsive to chilling stress, which mitigates the negative impacts of chilling through reducing reactive oxygen species content and enhancing cell membrane stability at the physiological and biochemical levels. We also found that the ZmPIF6 overexpression lines showed a significant increase in grain size, encompassing both length and width, which mainly due to the increase in cell size. In addition, digital gene expression results suggested that ZmPIF6 regulates the expression of cold-related and grain size-related genes in rice. In light of these findings, ZmPIF6 has a hopeful prospect as a candidate gene of chilling tolerance and crop productivity in the transgenic breeding.

PagKNAT2/6b regulates tension wood formation and gravitropism by targeting cytokinin metabolism.

Tension wood is a specialized xylem tissue associated with gravitropism in angiosperm trees. However, few regulators of tension wood formation have been identified. The molecular mechanisms underpinning tension wood formation remain elusive. Here, we report that a Populus KNOTTED-like homeobox gene, PagKNAT2/6b, is involved in tension wood formation and gravity response. Transgenic poplar plants overexpressing PagKNAT2/6b displayed more sensitive gravitropism than controls, as indicated by increased stem curvature. Microscopic examination revealed greater abundance of fibre cells with a gelatinous cell wall layer (G-layer) and asymmetric growth of secondary xylem in PagKNAT2/6b overexpression lines. Conversely, PagKNAT2/6b dominant repression plants exhibited decreased tension wood formation and reduced response to gravity stimulation. Moreover, sensitivity to gravity stimulation showed a negative relationship with development stage. Expression of genes related to growth and senescence was affected in PagKNAT2/6b transgenic plants. More importantly, transcription activation and electrophoretic mobility shift assays suggested that PagKNAT2/6b promotes the expression of cytokinin metabolism genes. Consistently, cytokinin content was increased in PagKNAT2/6b overexpression plants. Therefore, PagKNAT2/6b is involved in gravitropism and tension wood formation, likely via modulation of cytokinin metabolism.

O-Glycosyltransferase Gene BnaC09.OGT Involved in Regulation of Unsaturated Fatty Acid Biosynthesis for Enhancing Osmotic Stress Tolerance in Brassica napus L.

Osmotic stress is a major threaten to the growth and yield stability of Brassica napus. Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) is ubiquitous in plants, and participates in a variety of signal transduction and metabolic regulation. However, studies on the role of O-GlcNAc transferase (OGT) in osmotic stress tolerance of plants are limited. In previous study, a O-glycosyltransferase, named BnaC09.OGT, was identified from the B. napus variety 'Zhongshuang 11' by yeast one hybrid with promoter of BnaA01.GPAT9. It was found that BnaC09.OGT localized in both nucleus and cytoplasm. The spatiotemporal expression pattern of BnaC09.OGT exhibited tissue specificity in developmental seed, especially in 15 days after pollination. In view of osmotic stress inducing, the BnaC09.OGT overexpression and knockout transgenic lines were constructed for biological function study. Phenotypic analysis of BnaC09.OGT overexpression seedlings demonstrated that BnaC09.OGT could enhance osmotic stress tolerance than WT and knockout lines in euphylla stage under 15% PEG6000 treatment after 7 days. In addition, compared with WT and knockout lines, overexpression of BnaC09.OGT had significantly higher activities of antioxidant enzymes (SOD and POD), higher content of soluble saccharide, and while significantly less content of malondialdehyde, proline and anthocyanidin under 15% PEG6000 treatment after 7 days. On the other hand, the unsaturated fatty acid content of BnaC09.OGT overexpression was significantly higher than that of WT and knockout lines, so it is speculated that the BnaC09.OGT could increase unsaturated fatty acid biosynthesis for osmotic stress tolerance by promoting the expression of BnaA01.GPAT9 in glycerolipid biosynthesis. In summary, the above results revealed that the function of BnaC09.OGT provides new insight for the analysis of the pathway of O-glycosylation in regulating osmotic stress tolerance in B. napus.

Expression and antiviral application of exogenous lectin (griffithsin) in sweetpotatoes.

Griffithsin (GRFT) is a highly effective, broad-spectrum, safe, and stable viral inhibitor used to suppress a variety of viruses. However, little information is available on whether GRFT can prevent plant viral diseases. In this study, we constructed a GRFT overexpression vector containing the sweetpotato storage cell signal peptide and generated exogenous GRFT overexpression lines through genetic transformation. The transgenic plants showed notable resistance to sweetpotato virus disease in the virus nursery. To verify the antiplant virus function of GRFT, transient expression in tobacco leaves showed that GRFT inhibited the sweetpotato leaf curl virus (SPLCV). The replication of SPLCV was entirely inhibited when the concentration of GRFT reached a certain level. The results of pulldown and BIFC assays showed that GRFT did not interact with the six components of SPLCV. In addition, the mutated GRFTD/A without the binding ability of carbohydrate and anticoronavirus function, in which three aspartate residues at carbohydrate binding sites were all mutated to alanine, also inhibited SPLCV. Quantitative reverse-transcription PCR analyses showed that the tobacco antiviral-related genes HIN1, ICS1, WRKY40, and PR10 were overexpressed after GRFT/GRFTD/A injection. Furthermore, HIN1, ICS1, and PR10 were more highly expressed in the leaves injected with GRFTD/A. The results suggest that sweetpotato is able to express GRFT exogenously as a bioreactor. Moreover, exogenous GRFT expression inhibits plant viruses by promoting the expression of plant antiviral genes.

Receptor-like cytoplasmic kinase 58 reduces tolerance of maize seedlings to low magnesium via promoting H2O2 over-accumulation.

ZmRLCK58, a negative growth regulator, reduces tolerance of maize seedlings to low Mg via enhancing H2O2 accumulation in the shoot. Magnesium (Mg) deficiency is one of critical limiting factors for crop production in widespread acidic soils worldwide. However, the molecular mechanism of crop response to Mg deficiency is still largely unclear. Here, we found higher concentrations of H2O2, soluble sugars, and starch (1.5-, 1.9-, and 1.4-fold, respectively) in the shoot of low-Mg-treated maize seedlings, compared with Mg sufficient plants under hydroponic culture. Consistent with over-accumulation of H2O2, transcriptome profiling revealed significant enrichment of 175 differentially expressed genes (DEGs) in "response to oxygen-containing compound" out of 641 DEGs in the shoot under low Mg. Among 175 DEGs, a down-regulated receptor-like cytoplasmic kinase ZmRLCK58 underwent a recent duplication event before Poaceae divergence and was highly expressed in the maize shoot. ZmRLCK58 overexpression enhanced H2O2 accumulation in shoots by 21.3% and 29.8% under control and low-Mg conditions, respectively, while reducing biomass accumulation compared with wild-type plants. Low Mg further led to 39.7% less starch accumulation in the ZmRLCK58 overexpression shoot and lower Mg utilization efficiency. Compared with wild-type plants, overall down-regulated expression of genes related to response to carbohydrate, photosynthesis, H2O2 metabolic, oxidation-reduction, and ROS metabolic processes in ZmRLCK58 overexpression lines preconditioned aforementioned physiological alterations. Together, ZmRLCK58, as a negative growth regulator, reduces tolerance of maize seedlings to low Mg via enhancing H2O2 accumulation.