Transgenic Analysis Research Articles

MdKCS2 increased plant drought resistance by regulating wax biosynthesis

We found that the apple wax related gene played a role in changing plant epidermal permeability and enhancing plant resistance to drought stress by increasing wax accumulation. The content and composition of epidermal wax in plants are affected by genetic and environmental factors. The KCS gene encodes the β-ketoalionyl-CoA synthetase, which is a rate-limiting enzyme in the synthesis of very-long-chain fatty acids (VLCFAs). In this study, we identified the MdKCS2 gene from apple as a homolog of Arabidopsis AtKCS2. The KCS protein is localized on the endoplasmic reticulum membrane. MdKCS2 exhibited the highest expression in apple pericarp, and was induced by abiotic stresses, such as drought and salt. Transgenic analysis indicated that the MdKCS2 improved the resistance to abiotic stress in apple calli. Ectopic expression of MdKCS2 in Arabidopsis increased the content of wax in leaves and stems, changed the permeability of cuticle of leaves, and enhanced plant drought resistance.

Characterization on the P-associated and agronomic traits as well as associated molecular processes in wheat under Pi deprivation condition

Phosphorus (P) acts as one of the essential macronutrients and plays critical roles in regulating growth, development, and yield formation capacity of the crop plants. Elucidating the physiological and molecular processes underlying plant P deprivation responses benefit the crop cultivation with high P use efficiency (PUE). In this study, we investigated the P-associated and agronomic traits as well as the transcriptome profile under contrasting inorganic phosphorus (Pi) levels combined with deficit irrigation, using two wheat cultivars contrasted by PUE (i.e., high PUE Shixin 828 and P deprivation sensitive Jimai 518). The deficient-P (DP) treatment decreased P accumulative amounts, photosynthetic function, and biomass in plants at various growth stages and reduced yields with respect to sufficient-P (SP) condition. Although the two cultivars were comparable on growth and P-associated traits as well as yields under SP, Shixin 828 displayed better growth traits, more P accumulation, and higher yields than Jimai 518 under DP, suggesting that the high PUE cultivars with enhanced P uptake positively affects photosynthetic function, biomass production, and productivity of plants under P deprivation conditions. A large quantity of genes with differential expression patterns, including 2948 upregulated and 1844 downregulated, were identified based on RNA-seq analysis in the Shixin 828 plants treated with P deprivation. These DE genes were shown to be associated with biological process (i.e., metabolic process and cellular process etc.), cellular components (cell body and organelle etc.), and molecular function (binding and catalytic activity), and phytohormone signaling pathways. Transgene analysis on TaZFP1, a gene in ZFP transcription factor family that exhibited upregulated expression in P-deprived plants, validated its role in enhancing P accumulation and P starvation adaptation of plants. Our results suggested that plant P deprivation response is underlying modulation of various physiological processes via modifying gene transcription at global level.

Overexpression of a Banana Aquaporin Gene MaPIP1;1 Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Banana and Analysis of Its Interacting Transcription Factors.

Aquaporins can improve the ability of plants to resist abiotic stresses, but the mechanism is still not completely clear. In this research, overexpression of MaPIP1;1 in banana improved tolerance to multiple stresses. The transgenic plants resulted in lower ion leakage and malondialdehyde content, while the proline, chlorophyll, soluble sugar, and abscisic acid (ABA) contents were higher. In addition, under high salt and recovery conditions, the content of Na+ and K+ is higher, also under recovery conditions, the ratio of K+/Na+ is higher. Finally, under stress conditions, the expression levels of ABA biosynthesis and response genes in the transgenic lines are higher than those of the wild type. In previous studies, we proved that the MaMADS3 could bind to the promoter region of MaPIP1;1, thereby regulating the expression of MaPIP1;1 and affecting the drought tolerance of banana plants. However, the mechanism of MaPIP1;1 gene response to stress under different adversity conditions might be regulated differently. In this study, we proved that some transcription factor genes, including MaERF14, MaDREB1G, MaMYB1R1, MaERF1/39, MabZIP53, and MaMYB22, showed similar expression patterns with MaPIP1;1 under salt or cold stresses, and their encoded proteins could bind to the promoter region of MaPIP1;1. Here we proposed a novel MaPIP1;1-mediated mechanism that enhanced salt and cold tolerance in bananas. The results of this study have enriched the stress-resistant regulatory network of aquaporins genes and are of great significance for the development of molecular breeding strategies for stress-resistant fruit crops.

Transcriptome analysis reveals abscisic acid enhancing drought resistance by regulating genes related to flavonoid metabolism in pigeon pea

Abscisic acid (ABA) plays an important role in drought resistance of plants. Plants subjected to drought stress often produce a large number of secondary metabolites. More and more studies indicate that secondary metabolites are directly related to plant stress resistance. However, there are few reports about the influence of ABA regulating secondary metabolism on drought. In our study, we found that as a pioneer tree species in karst landforms, the drought resistance can also be improved by ABA. ABA can reduce the stomatal opening of pigeon pea under drought stress and reduce the influx of H+ from the root tip. At the same time, we found that in ABA-treated pigeon pea, flavonoid metabolites accumulate in large amounts, especially genistein, genistin and pterostilbene. Through transcriptome sequencing analysis, it was found that after ABA treatment, flavonoids biosynthesis pathways changed significantly; among them, the key enzyme genes CHS, HIDH and ROMT in the biosynthesis pathway of genistein, genistin and pterostilbene were significantly up-regulated. According to the co-expression analysis in the transcriptome data, we screened that multiple MYB and bHLH transcription factors may be involved in the regulation of flavonoid accumulation. And the biosynthetic genes contained binding sites of related transcription factors. Through RT-PCR, we further analyzed the expression of MYB, bHLH and related biosynthetic genes after ABA treatment at different times, and found that they were all regulated by ABA. By High Performance Liquid Chromatography (HPLC) analysis, we found the increase of genistein, genistin and pterostilbene content in pigeon pea treated with ABA under drought stress. Through hairy root transgenic analysis, We found that CcMYB114 regulate drought resistance by modulating the expression of CcHIDH1 and CcHIDH3, and then regulating genistein and genistin accumulation in pigeon pea. This study provides evidence to analyze the regulation of ABA in pigeon pea on the metabolism of flavonoids and thus affect its drought resistance.

Functional homoeologous alleles of CONSTANS contribute to seasonal crop type in rapeseed.

Two CO paralogs in Brassica napus were confirmed and shown distinct expression pattern and function in promoting flowering and allelic variation s within BnaCO.A10 were found closely associated with ecotype divergence. CONSTANS (CO) is a key gene that responds to photoperiod and in Arabidopsis can promote flowering under long-day (LD) conditions. Brassica napus L. is a major oil crop and close relative of Arabidopsis, and arose via allopolyploidization from the diploids B. rapa (A genome) and B. oleracea (C genome). In this study, we confirmed that B. napus has two CO genes located on the A10 (BnaCO.A10) and C9 (BnaCO.C9) chromosomes. Significant differences in level and temporal pattern of transcription, as well as in protein function, of these homoeologous may have resulted from sequence variation in the promoter as well as in the coding region. Apart from two insertions of 527bp and 2002bp in the promoter of BnaCO.C9 that function as transcriptional enhancers, this gene is otherwise highly conserved in both promoter and coding region. However, BnaCO.A10 was classified into two haplotypes and transgene analysis in Arabidopsis and backcross analysis in rapeseed indicated that the winter-type haplotype had a greater effect in promoting flowering than the spring type. We discuss the contribution of CO alleles to species evolution, and for eco-geographic radiation following crop domestication, alongside scope for managing this locus in future breeding.

Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

Coumarin Interferes with Polar Auxin Transport Altering Microtubule Cortical Array Organization in Arabidopsis thaliana (L.) Heynh. Root Apical Meristem.

Coumarin is a phytotoxic natural compound able to affect plant growth and development. Previous studies have demonstrated that this molecule at low concentrations (100 µM) can reduce primary root growth and stimulate lateral root formation, suggesting an auxin-like activity. In the present study, we evaluated coumarin’s effects (used at lateral root-stimulating concentrations) on the root apical meristem and polar auxin transport to identify its potential mode of action through a confocal microscopy approach. To achieve this goal, we used several Arabidopsis thaliana GFP transgenic lines (for polar auxin transport evaluation), immunolabeling techniques (for imaging cortical microtubules), and GC-MS analysis (for auxin quantification). The results highlighted that coumarin induced cyclin B accumulation, which altered the microtubule cortical array organization and, consequently, the root apical meristem architecture. Such alterations reduced the basipetal transport of auxin to the apical root apical meristem, inducing its accumulation in the maturation zone and stimulating lateral root formation.

Functional Relationship of Arabidopsis AOXs and PTOX Revealed via Transgenic Analysis.

Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.

Integrated global analysis reveals a vitamin E-vitamin K1 sub-network, downstream of COLD1, underlying rice chilling tolerance divergence.

Rice, a staple food with tropical/subtropical origination, is susceptible to cold stress, one of the major constraints on its yield and distribution. Asian cultivated rice consists of two subspecies with diverged chilling tolerance to adapt to different environments. The mechanism underlying this divergence remains obscure with a few known factors, including membrane protein CHILLING-TOLERANCE DIVERGENCE 1 (COLD1). Here, we reveal a vitamin E-vitamin K1 sub-network responsible for chilling tolerance divergence through global analyses. Rice genome regions responsible for tolerance divergence are identified with chromosome segment substitution lines (CSSLs). Comparative transcriptomic and metabolomic analysis of chilling-tolerant CSSL4-1 and parent lines uncovered a vitamin E-vitamin K1 sub-network in chloroplast with tocopherol (vitamin E) mediating chloroplast-to-nucleus signaling. COLD1, located in the substitution segment in CSSL4-1, is confirmed as its upstream regulator by transgenic material analysis. Our work uncovers a pathway downstream of COLD1, through which rice modulates chilling tolerance for thermal adaptation, with potential utility in crop improvement.

The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (−1183 to −988 and −2018 to −1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

Revisiting the consequences of deleting the X inactivation center

Mammalian cells equalize X-linked dosages between the male (XY) and female (XX) sexes by silencing one X chromosome in the female sex. This process, known as "X chromosome inactivation" (XCI), requires a master switch within the X inactivation center (Xic). The Xic spans several hundred kilobases in the mouse and includes a number of regulatory noncoding genes that produce functional transcripts. Over three decades, transgenic and deletional analyses have demonstrated both the necessity and sufficiency of the Xic to induce XCI, including the steps of X chromosome counting, choice, and initiation of whole-chromosome silencing. One recent study, however, reported that deleting the noncoding sequences of the Xic surprisingly had no effect for XCI and attributed a sufficiency to drive counting to the coding gene, Rnf12/Rlim Here, we revisit the question by creating independent Xic deletion cell lines. Multiple independent clones carrying heterozygous deletions of the Xic display an inability to up-regulate Xist expression, consistent with a counting defect. This defect is rescued by a second site mutation in Tsix occurring in trans, bypassing the defect in counting. These findings reaffirm the essential nature of noncoding Xic elements for the initiation of XCI.

Omics and Transgenic Analyses Reveal that Salvianolic Acid B Exhibits its Anti-Inflammatory Effects through Inhibiting the Mincle-Syk-Related Pathway in Macrophages.

Salvianolic acid B (Sal B), the main water-soluble compound in Salvia miltiorrhiza, is known to exhibit anti-inflammatory activity, however, the underlying mechanism(s) is not completely uncovered. In this study, Sal B inhibited lipopolysaccharide (LPS)-induced M1 activation and promoted the transformation of macrophages from M1- to M2-type polarization. The altered lipid profiles of LPS-induced RAW 264.7 macrophages were partly restored by Sal B treatment. At the proteomic level, a total of 5612 proteins were identified and 432 were significantly changed in macrophages under LPS treatment. The differential proteins were classified into four clusters according to their expression level in blank, LPS, and Sal B groups. LPS-induced proteins in Cluster IV including Kif14, Mincle, and Sec62 were significantly recovered to almost normal levels by Sal B treatment. Use of knockdown Mincle or picetannol (inhibitor of Syk) led to significant reductions in the gene expressions of IL-1β, iNOS, and IL-12 and the release of NO. The converse was, however, observed for overexpressed Mincle. In addition, LPS- or trehalose-6,6-dibehenate-induced phosphorylation of Syk and PKCδ was decreased by Sal B treatment. These results suggest that Sal B inhibition of LPS-induced inflammation might be through inhibition of the Mincle-Syk-PKCδ signaling pathway.

English

Improving potassium (K) use efficiency (KUE) is beneficial for the sustainable production of cereal crops. In this study, the effects of K input level on its uptake and agronomic trait of the winter wheat under deficit irrigation were investigated in K deprivation responses, using two cultivars contrasting (low-K tolerant cultivar Kenong 9204 and K deprivation sensitive one Jimai 120). Under sufficient-K treatment (K180, SK), the two cultivars showed similar K contents, and K accumulation, biomass, photosynthetic parameters in upper expanded leaves, including yield components. Under deficient-K (K60, DK) condition, both cultivars showed varied behaviors of the K-associated traits, physiological parameters, growth and agronomic traits; however, better response was observed in Kenong 9204 than Jimai 120. These results suggested the essential roles of low-K tolerant cultivars under the K-saving management together with deficit irrigation. Two genes of the potassium transporter (HAK) family, TaHAK3 and TaHAK5, showed expression of significantly upregulated upon K deprivation, with much more transcripts shown in the K-deprived Kenong 9204 plants than Jimai 120 ones. Transgene analysis on the HAK genes validated their positive roles in modulating the K accumulation and biomass production of plants under low-K condition. These results indicated that distinct HAK family genes are transcriptionally regulated underlying K deprivation signaling and contribute to plant K uptake and biomass production under low-K conditions. This study suggested the drastically genetic variation on K uptake and biomass production across winter wheat cultivars treated by K- and water-saving conditions, associated with transcription efficiency of the distinct HAK genes which modulate K uptake, growth and development of plants. © 2021 Friends Science Publishers

O-linked N-acetylglucosamine transferase is involved in fine regulation of flowering time in winter wheat

Vernalization genes underlying dramatic differences in flowering time between spring wheat and winter wheat have been studied extensively, but little is known about genes that regulate subtler differences in flowering time among winter wheat cultivars, which account for approximately 75% of wheat grown worldwide. Here, we identify a gene encoding an O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) that differentiates heading date between winter wheat cultivars Duster and Billings. We clone this TaOGT1 gene from a quantitative trait locus (QTL) for heading date in a mapping population derived from these two bread wheat cultivars and analyzed in various environments. Transgenic complementation analysis shows that constitutive overexpression of TaOGT1b from Billings accelerates the heading of transgenic Duster plants. TaOGT1 is able to transfer an O-GlcNAc group to wheat protein TaGRP2. Our findings establish important roles for TaOGT1 in winter wheat in adaptation to global warming in the future climate scenarios.

Hemodynamic Forces Regulate Cardiac Regeneration-Responsive Enhancer Activity during Ventricle Regeneration.

Cardiac regenerative capacity varies widely among vertebrates. Zebrafish can robustly regenerate injured hearts and are excellent models to study the mechanisms of heart regeneration. Recent studies have shown that enhancers are able to respond to injury and regulate the regeneration process. However, the mechanisms to activate these regeneration-responsive enhancers (RREs) remain poorly understood. Here, we utilized transient and transgenic analysis combined with a larval zebrafish ventricle ablation model to explore the activation and regulation of a representative RRE. lepb-linked enhancer sequence (LEN) directed enhanced green fluorescent protein (EGFP) expression in response to larval ventricle regeneration and such activation was attenuated by hemodynamic force alteration and mechanosensation pathway modulation. Further analysis revealed that Notch signaling influenced the endocardial LEN activity as well as endogenous lepb expression. Altogether, our work has established zebrafish models for rapid characterization of cardiac RREs in vivo and provides novel insights on the regulation of LEN by hemodynamic forces and other signaling pathways during heart regeneration.

Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained. Southern blot, fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and, with the exception of mating, give ambiguous results. With the emergence of the real-time quantitative PCR technology, different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis. However, the accuracy in quantitation by this method can be influenced in all steps of analysis. This review focuses on the aspects that influence pre-analytical steps (DNA extraction and DNA quantification methods), quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.

Identification of transcription factors controlling cell wall invertase gene expression for reproductive development via bioinformatic and transgenic analyses.

Cell wall invertase (CWIN) hydrolyses sucrose into glucose and fructose in the extracellular matrix and plays crucial roles in assimilate partitioning and sugar signalling. However, the molecular regulators controlling CWIN gene transcription remain unknown. As the first step to address this issue, we performed bioinformatic and transgenic studies, which identified a cohort of transcription factors (TFs) modulating CWIN gene expression in Arabidopsis thaliana. Comprehensive bioinformatic analyses identified 18 TFs as putative regulators of the expression of AtCWIN2 and AtCWIN4 that are predominantly expressed in Arabidopsis reproductive organs. Among them, MYB21, ARF6, ARF8, AP3 and CRC were subsequently shown to be the most likely regulators of CWIN gene expression based on molecular characterization of the respective mutant of each candidate TF. More specifically, the obtained data indicate that ARF6, ARF8 and MYB21 regulate CWIN2 expression in the anthers and CWIN4 in nectaries, anthers and petals, whereas AP3 and CRC were determined primarily to regulate the transcriptional activity of CWIN4. TF-promoter interaction assays demonstrated that ARF6 and ARF8 directly control CWIN2 and CWIN4 transcription with AP3 activating CWIN4. The involvement of ARF8 in regulating CWIN4 expression was further supported by the finding that enhanced CWIN4 expression partially recovered the short silique phenotype displayed by the arf8-3 mutant. The identification of the five TFs regulating CWIN expression serves as a launching pad for future studies to dissect the upstream molecular network underpinning the transcription of CWINs and provides a new avenue, potentially, to engineer assimilate allocation and reproductive development for improving seed yield.

Nitrogen partitioning traits and expression patterns of N metabolism‐associated genes in maize hybrids with contrasting N utilization efficiencies

AbstractThe nitrogen (N) uptake and utilization in maize (Zea mays L.) plants are accomplished through a series of biochemical reactions and transformations involving the N assimilation enzymes. Nitrate reductase (NR) and glutamine synthetase (GS) exert important functions in the N assimilation and remobilization processes. The mechanisms underlying plant N partitioning and the associated expression patterns of NR and GS genes in maize hybrids with different N utilization efficiencies were still not clear. In this study, the expression of ZmNR and ZmGS genes that are related to N partitioning were analyzed in two maize hybrids (XY335 and HN138). Results showed that XY335 displayed higher grain yield, kernel numbers, shoot N uptake, and physiological N utilization efficiency (NUtE) than those shown in HN138 under low‐N conditions. The higher post‐silking N uptake and N remobilization in vegetative organs in XY335 were associated with the enhanced net photosynthetic rate (Pn), the NR and GS activities in ear‐leaf. Additionally, the expression levels of ZmGS1.3 and ZmGS1.4 were up‐regulated when exposed to low‐N stress, showing higher values in XY335 than those in HN138. Correlation analysis showed that the expression levels of ZmNR, ZmGS1.3, and ZmGS1.4 are correlated with leaf NR and GS activities, N remobilization, and grain yield. Our investigation suggested the critical roles of the N metabolism‐associated genes in the modulation of internal N translocation and plant productivity in maize. Future transgene analysis on ZmNR and ZmGS can provide insights into the molecular mechanisms underlying plant tolerance to low N stress in maize.

Characterization on the physiological traits of plants and yield formation capacity upon water- and N-saving conditions in wheat (T. aestivum L.)

Improving water and nitrogen (N) use efficiencies of the Triticum aestivum species contributes to the sustainable agriculture worldwide. In this study, we investigated the physiological traits of plants and the yield formation capacity of winter wheat treated with deficit irrigation together with N-saving condition. Compared with OI-ON (optimum irrigation, OI together with optimum nitrogen, ON), treatment DI-DN (deficit irrigation, DI combined with deficient N (DN, N120) led to decreased contents of chlorophyll, nitrogen (N), and osmolytes, Pn, and the activities of SOD and POD in leaves at various stages together with reduced yields in Jimai 585 and Shimai 22, two cultivars with contrast drought stress responses. The moisture contents in soil profile planted by tested cultivars showed significant reduction under OI-ON with respect to those under DI-DN, suggesting the positive impact of elevated soil water consumption on plant physiological and agronomic traits. The drought-tolerant cultivar Shimai 22 displayed much more improvement on the physiological and agronomic traits, WUE, and NUE than drought-sensitive cultivar Jimai 585 under DI-DN, due to effective usage for soil water and N storage. Yields and grain weights under DI-DN were positively correlated with chlorophyll and osmolytes contents, Pn, and antioxidant enzyme activities, suggesting that the improved physiological traits under DI-DN contributed to the elevated yield for the drought-tolerant cultivar. Two nitrate transporter (NRT) family ones TaNRT2.1 and TaNRT2.2 and TaPYR3 and TaPYR6, two abscisic acid receptor (PYR) family genes exhibited induced transcripts upon DI-DN in wheat cultivars, with more abundance in Shimai 22 than in Jimai 585. Transgenic analysis on the differential genes confirmed their functions in mediating plant drought and N deprivation responses, suggesting that distinct PYR and NRT family members mediate drought and N deprivation adaptation in wheat plants. Our investigation provided insights into high WUE and NUE cultivation of winter wheat under the limited-water and N-saving management.

Genome-wide identification and characterization of long non-coding RNAs involved in flag leaf senescence of rice

Key messageThis study showed the systematic identification of long non-coding RNAs (lncRNAs) involving in flag leaf senescence of rice, providing the possible lncRNA-mRNA regulatory relationships and lncRNA-miRNA-mRNA ceRNA networks during leaf senescence.LncRNAs have been reported to play crucial roles in diverse biological processes. However, no systematic identification of lncRNAs associated with leaf senescence in plants has been studied. In this study, a genome-wide high throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. A total of 3953 lncRNAs and 38757 mRNAs were identified, of which 343 lncRNAs and 9412 mRNAs were differentially expressed. Through weighted gene co-expression network analysis (WGCNA), 22 continuously down-expressed lncRNAs targeting 812 co-expressed mRNAs and 48 continuously up-expressed lncRNAs targeting 1209 co-expressed mRNAs were considered to be significantly associated with flag leaf senescence. Gene Ontology results suggested that the senescence-associated lncRNAs targeted mRNAs involving in many biological processes, including transcription, hormone response, oxidation–reduction process and substance metabolism. Additionally, 43 senescence-associated lncRNAs were predicted to target 111 co-expressed transcription factors. Interestingly, 8 down-expressed lncRNAs and 29 up-expressed lncRNAs were found to separately target 12 and 20 well-studied senescence-associated genes (SAGs). Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 6 down-expressed lncRNAs possibly regulated 51 co-expressed mRNAs through 15 miRNAs, and 14 up-expressed lncRNAs possibly regulated 117 co-expressed mRNAs through 21 miRNAs. Importantly, by expression validation, a conserved miR164-NAC regulatory pathway was found to be possibly involved in leaf senescence, where lncRNA MSTRG.62092.1 may serve as a ceRNA binding with miR164a and miR164e to regulate three transcription factors. And two key lncRNAs MSTRG.31014.21 and MSTRG.31014.36 also could regulate the abscisic-acid biosynthetic gene BGIOSGA025169 (OsNCED4) and BGIOSGA016313 (NAC family) through osa-miR5809. The possible regulation networks of lncRNAs involving in leaf senescence were discussed, and several candidate lncRNAs were recommended for prior transgenic analysis. These findings will extend the understanding on the regulatory roles of lncRNAs in leaf senescence, and lay a foundation for functional research on candidate lncRNAs.