Developmental pleiotropy of SDP1 from seedling to mature stages in B. napus.

Rapeseed, an important oil crop, relies on robust seedling emergence for optimal yields. Seedling emergence in the field is vulnerable to various factors, among which inadequate self-supply of energy is crucial to limiting seedling growth in early stage. SUGAR-DEPENDENT1 (SDP1) initiates triacylglycerol (TAG) degradation, yet its detailed function has not been determined in B. napus. Here, we focused on the effects of plant growth during whole growth stages and energy mobilization during seedling establishment by mutation in BnSDP1. Protein sequence alignment and haplotypic analysis revealed the conservation of SDP1 among species, with a favorable haplotype enhancing oil content. Investigation of agronomic traits indicated bnsdp1 had a minor impact on vegetative growth and no obvious developmental defects when compared with wild type (WT) across growth stages. The seed oil content was improved by 2.0-2.37% in bnsdp1 lines, with slight reductions in silique length and seed number per silique. Furthermore, bnsdp1 resulted in lower seedling emergence, characterized by a shrunken hypocotyl and poor photosynthetic capacity in the early stages. Additionally, impaired seedling growth, especially in yellow seedlings, was not fully rescued in medium supplemented with exogenous sucrose. The limited lipid turnover in bnsdp1 was accompanied by induced amino acid degradation and PPDK-dependent gluconeogenesis pathway. Analysis of the metabolites in cotyledons revealed active amino acid metabolism and suppressed lipid degradation, consistent with the RNA-seq results. Finally, we proposed strategies for applying BnSDP1 in molecular breeding. Our study provides theoretical guidance for understanding trade-off between oil accumulation and seedling energy mobilization in B. napus.

Calcium regulation of the Arabidopsis Na+/K+ transporter HKT1;1 improves seed germination under salt stress.

Calcium is known to improve seed-germination rates under salt stress. We investigated the involvement of calcium ions (Ca2+) in regulating HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1; 1), which encodes a Na+/K+ transporter, and its post-translational regulator TYPE 2C PROTEIN PHOSPHATASE 49 (PP2C49), in germinating Arabidopsis (Arabidopsis thaliana) seedlings. Germination rates of hkt1 mutant seeds under salt stress remained unchanged by CaCl2 treatment in wild-type Arabidopsis, whereas pp2c49 mutant seeds displayed improved salt-stress tolerance in the absence of CaCl2 supplementation. Analysis of HKT1;1 and PP2C49 promoter activity revealed that CaCl2 treatment results in radicle-focused expression of HKT1;1 and reduction of the native radicle-exclusive expression of PP2C49. Ion-content analysis indicated that CaCl2 treatment improves K+ retention in germinating wild-type seedlings under salt stress, but not in hkt1 seedlings. Transgenic seedlings designed to exclusively express HKT1;1 in the radicle during germination displayed higher germination rates under salt stress than the wild type in the absence of CaCl2 treatment. Transcriptome analysis of germinating seedlings treated with CaCl2, NaCl, or both revealed 118 upregulated and 94 downregulated genes as responsive to the combined treatment. Bioinformatics analysis of the upstream sequences of CaCl2-NaCl-treatment-responsive upregulated genes revealed the abscisic acid response element CACGTGTC, a potential CaM-binding transcription activator-binding motif, as most prominent. Our findings suggest a key role for Ca2+ in mediating salt-stress responses during germination by regulating genes that function to maintain Na+ and K+ homeostasis, which is vital for seed germination under salt stress.

Integration of mRNA and miRNA analysis reveals the molecular mechanisms of sugar beet (Beta vulgaris L.) response to salt stress.

The continuous increase of saline-alkali areas worldwide has led to the emergence of saline-alkali conditions, which are the primary abiotic stress or hindering the growth of plants. Beet is among the main sources of sugar, and its yield and sugar content are notably affected by saline-alkali stress. Despite sugar beet being known as a salt-tolerant crop, there are few studies on the mechanisms underlying its salt tolerance, and previous studies have mainly delineated the crop's response to stress induced by NaCl. Recently, advancements in miRNA-mRNA network analysis have led to an increased understanding of how plants, including sugar beet, respond to stress. In this study, seedlings of beet variety "N98122" were grown in the laboratory using hydroponics culture and were exposed to salt stress at 40 days of growth. According to the phenotypic adaptation of the seedlings' leaves from a state of turgidity to wilting and then back to turgidity before and after exposure, 18 different time points were selected to collect samples for analysis. Subsequently, based on the data of real-time quantitative PCR (qRT-PCR) of salt-responsive genes, the samples collected at the 0, 2.5, 7.5, and 16 h time points were subjected to further analysis with experimental materials. Next, mRNA-seq data led to the identification of 8455 differentially expressed mRNAs (DEMs) under exposure to salt stress. In addition, miRNA-seq based investigation retrieved 3558 miRNAs under exposure to salt stress, encompassing 887 known miRNAs belonging to 783 families and 2,671 novel miRNAs. With the integrated analysis of miRNA-mRNA network, 57 miRNA-target gene pairs were obtained, consisting of 55 DEMIs and 57 DEMs. Afterwards, we determined the pivotal involvement of aldh2b7, thic, and δ-oat genes in the response of sugar beet to the effect of salt stress. Subsequently, we identified the miRNAs novel-m035-5p and novel-m0365-5p regulating the aldh gene and miRNA novel-m0979-3p regulating the thic gene. The findings of miRNA and mRNA expression were validated by qRT-PCR.

Integrated Analysis of Metabolome and Transcriptome Reveals Insights for Low Phosphorus Tolerance in Wheat Seedling.

Low phosphorus (LP) stress leads to a significant reduction in wheat yield, primarily in the reduction of biomass, the number of tillers and spike grains, the delay in heading and flowering, and the inhibition of starch synthesis and grouting. However, the differences in regulatory pathway responses to low phosphorus stress among different wheat genotypes are still largely unknown. In this study, metabolome and transcriptome analyses of G28 (LP-tolerant) and L143 (LP-sensitive) wheat varieties after 72 h of normal phosphorus (CK) and LP stress were performed. A total of 181 and 163 differentially accumulated metabolites (DAMs) were detected for G28CK vs. G28LP and L143CK vs. L143LP, respectively. Notably, the expression of pilocarpine (C07474) in G28CK vs. G28LP was significantly downregulated 4.77-fold, while the expression of neochlorogenic acid (C17147) in L143CK vs. L143LP was significantly upregulated 2.34-fold. A total of 4023 differentially expressed genes (DEGs) were acquired between G28 and L143, of which 1120 DEGs were considered as the core DEGs of LP tolerance of wheat after LP treatment. The integration of metabolomics and transcriptomic data further revealed that the LP tolerance of wheat was closely related to 15 metabolites and 18 key genes in the sugar and amino acid metabolism pathway. The oxidative phosphorylation pathway was enriched to four ATPases, two cytochrome c reductase genes, and fumaric acid under LP treatment. Moreover, PHT1;1, TFs (ARFA, WRKY40, MYB4, MYB85), and IAA20 genes were related to the Pi starvation stress of wheat roots. Therefore, the differences in LP tolerance of different wheat varieties were related to energy metabolism, amino acid metabolism, phytohormones, and PHT proteins, and precisely regulated by the levels of various molecular pathways to adapt to Pi starvation stress. Taken together, this study may help to reveal the complex regulatory process of wheat adaptation to Pi starvation and provide new genetic clues for further study on improving plant Pi utilization efficiency.

Exploring root system architecture and anatomical variability in alfalfa (Medicago sativa L.) seedlings.

BACKGROUND: The growth of alfalfa (Medicago sativa L.) is significantly hampered by drought and nutrient deficiencies. The identification of root architectural and anatomical characteristics holds paramount importance for the development of alfalfa genotypes with enhanced adaptation to adverse environmental conditions. In this study, we employed a visual rhizobox system to investigate the variability in root system architecture (including root depth, root length, root tips number, etc.), anatomical features (such as cortical traits, total stele area, number and area of vessel, etc.), as well as nitrogen and phosphorus uptake across 53 alfalfa genotypes during the seedling stage. RESULTS: Out of the 42 traits measured, 21 root traits, along with nitrogen (N) and phosphorus (P) uptake, displayed higher coefficients of variation (CVs ≥ 0.25) among the tested genotypes. Local root morphological and anatomical traits exhibited more significant variation than global root traits. Twenty-three traits with CVs ≥ 0.25 constituted to six principal components (eigenvalues > 1), collectively accounting for 88.0% of the overall genotypic variation. Traits such as total root length, number of root tips, maximal root depth, and others exhibited positive correlations with shoot dry mass and root dry mass. Additionally, total stele area and xylem vessel area showed positive correlations with N and P uptake. CONCLUSIONS: These root traits, which have demonstrated associations with biomass and nutrient uptake, may be considered for the breeding of alfalfa genotypes that possess efficient resource absorption and increased adaptability to abiotic stress, following validation during the entire growth period in the field.

Transcriptome analysis reveals the molecular mechanism of different forms of selenium in reducing cadmium uptake and accumulation in wheat seedlings.

Selenium (Se) can counteract cadmium (Cd) toxicity in wheat, but the molecular mechanism of different Se forms reducing Cd uptake and accumulation in wheat seedlings remain unclear. Here, a hydroponic experiment was conducted to investigate the effects of three Se forms (selenite (Se(IV)), selenate (Se(VI)) and seleno-L-methionine (SeMet)) on Cd2+ influx, Cd subcellular distribution, and Cd accumulation in wheat seedlings, and the underlying molecular mechanisms were investigated through transcriptome analysis. Consequently, Se(IV) and Se(VI) addition significantly reduced root Cd concentration by 74.3% and 80.8%, respectively, and all Se treatments significantly decreased shoot Cd concentration by approximately 34.2%-74.9%, with Se(IV) addition having the most pronounced reducing effect. Transcriptome analysis showed the reduction of Cd accumulation after Se(IV) addition was mainly due to the downregulation of Cd uptake genes. The inhibition of Cd accumulation after Se(VI) addition was not only associated with the downregulation of Cd uptake genes, but also related to the sequestration of Cd in vacuole. For SeMet addition, the reduction of Cd accumulation was mainly related to the sequestration of Cd in vacuole as GSH-Cd. The above findings provide novel insights to understand the effects of different forms of Se on Cd uptake and accumulation and tolerance in wheat.

Genetic diversity, plant growth promotion potential, and antimicrobial activity of culturable endophytic actinobacteria isolated from Aconitum carmichaelii Debeaux.

AIM: This study evaluated the phylogenetic diversity, plant growth promotion capacity, antifungal activity, and biocontrol potential of culturable actinobacterial endophytes isolated from the medicinal plant Aconitum carmichaelii Debeaux. METHODS AND RESULTS: Isolation of actinobacteria from healthy A. carmichaelii plants was carried out on six different media. Full-length 16S rRNA gene was amplified by PCR from the genomic DNA of each strain. Indole-3-acetic acid and siderophore production were quantitatively assessed by the Salkowski and Chrome Azurol S methods, respectively. Rice seeds germination and seedling growth were employed to evaluate plant growth promotion capacities of candidate strains. Dual-culture assay and pot experiments were performed to investigate the antifungal and biocontrol potential of isolates. We obtained 129 actinobacterial isolates from A. carmichaelii, and they belonged to 49 species in 7 genera. These strains exhibited diverse plant growth promotion ability, among which one strain significantly enhanced rice seeds germination, while 31 strains significantly facilitated rice seedling growth. SWUST-123 showed strong antifungal activity against four pathogens in vitro and was most compatible with Qingchuan cultivar. SWUST-123 reduced around 40% of southern blight disease occurrence compared to blank control treatment. . CONCLUSION: Aconitum carmichaelii harbored genetically diverse actinobacterial endophytes exhibiting diverse plant growth promotion and antifungal potential, some of which can be served as good candidates for biofertilizers and biocontrol agents.

Two types of GLR channels cooperate differently in light and dark growth of Arabidopsis seedlings.

BACKGROUND: GLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark. RESULTS: Wild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development. CONCLUSIONS: Our work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

Identification of core genes associated with different phosphorus levels in quinoa seedlings by weighted gene co-expression network analysis.

BACKGROUND: Quinoa is a highly nutritious and novel crop that is resistant to various abiotic stresses. However, its growth and development is restricted due to its limited utilization of soil phosphorus. Studies on the levels of phosphorus in quinoa seedlings are limited; therefore, we analyzed transcriptome data from quinoa seedlings treated with different concentrations of phosphorus. RESULTS: To identify core genes involved in responding to various phosphorus levels, the weighted gene co-expression network analysis method was applied. From the 12,085 expressed genes, an analysis of the gene co-expression network was done. dividing the expressed genes into a total of twenty-five different modules out of which two modules were strongly correlated with phosphorus levels. Subsequently we identified five core genes that correlated strongly either positively or negatively with the phosphorus levels. Gene ontology and assessments of the Kyoto Encyclopedia of Genes and Genomes have uncovered important biological processes and metabolic pathways that are involved in the phosphorus level response. CONCLUSIONS: We discovered crucial new core genes that encode proteins from various transcription factor families, such as MYB, WRKY, and ERF, which are crucial for abiotic stress resistance. This new library of candidate genes associated with the phosphorus level responses in quinoa seedlings will help in breeding varieties that are tolerant to phosphorus levels.

Genome skimming as an efficient tool for authenticating commercial products of the pharmaceutically important Paris yunnanensis (Melanthiaceae).

BACKGROUND: Paris yunnanensis (Melanthiaceae) is a traditional Chinese medicinal plant of significant pharmaceutical importance. Due to previous taxonomic confusion, a congeneric species, Paris liiana, has been mistaken for P. yunnanensis and cultivated on a large scale, leading to the mixing of commercial products (i.e., seedlings and processed rhizomes) of P. yunnanensis with those of P. liiana. This may have adverse effects on quality control in the standardization of P. yunnanensis productions. As the lack of PCR amplifiable genomic DNA within processed rhizomes is an intractable obstacle to the authentication of P. yunnanensis products using PCR-based diagnostic tools, this study aimed to develop a PCR-free method to authenticate commercial P. yunnanensis products, by applying genome skimming to generate complete plastomes and nrDNA arrays for use as the molecular tags. RESULTS: Based on a dense intraspecies sampling of P. liiana and P. yunnanensis, the robustness of the proposed authentication systems was evaluated by phylogenetic inferences and experimental authentication of commercial seedling and processed rhizome samples. The results indicate that the genetic criteria of both complete plastomes and nrDNA arrays were consistent with the species boundaries to achieve accurate discrimination of P. yunnanensis and P. liinna. Owing to its desirable accuracy and sensitivity, genome skimming can serve as an effective and sensitive tool for monitoring and controlling the trade of P. yunnanensis products. CONCLUSION: This study provides a new way to solve the long-standing problem of the molecular authentication of processed plant products due to the lack of PCR amplifiable genomic DNA. The proposed authentication system will support quality control in the standardization of P. yunnanensis products in cultivation and drug production. This study also provides molecular evidence to clarify the long-standing taxonomic confusion regarding the species delimitation of P. yunnanensis, which will contribute to the rational exploration and conservation of the species.

Lipid Metabolomic and Transcriptomic Analyses Reveal That Phosphatidylcholine Enhanced the Resistance of Peach Seedlings to Salt Stress through Phosphatidic Acid.

Soil salinity is a major conlinet limiting sustainable agricultural development in peach tree industry. In this study, lipid metabolomic pathway analysis indicated that phosphatidic acid is essential for root resistance to salt stress in peach seedlings. Through functional annotation analysis of differentially expressed genes in transcriptomics, we found that MAPK signaling pathway is closely related to peach tree resistance to salt stress, wherein PpMPK6 expression is significantly upregulated. Under salt conditions, the OE-PpMPK6 Arabidopsis thaliana (L.) Heynh. line showed higher resistance to salt stress than WT and KO-AtMPK6 lines. Furthermore, we found that the Na+ content in OE-PpMPK6 roots was significantly lower than that in WT and KO-AtMPK6 roots, indicating that phosphatidic acid combined with PpMPK6 activated the SOS1 (salt-overly-sensitive 1) protein to enhance Na+ efflux, thus alleviating the damage caused by NaCl in roots; these findings provide insight into the salt stress-associated transcriptional regulation.

Effects of different phosphorus levels on tiller bud development in hydroponic Phyllostachys edulis seedlings.

An appropriate amount of phosphate fertilizer can improve the germination rate of bamboo buds and increase the bamboo shoot output. However, the underlying biological mechanisms of phosphate fertilizer in bamboo shoot development have not been systematically reported. Herein, the effects of low (LP, 1 µM), normal (NP, 50 µM) and high (HP, 1000 µM) phosphorus (P) on the growth and development of moso bamboo (Phyllostachys edulis) tiller buds were first investigated. Phenotypically, the seedling biomass, average number of tiller buds and bud height growth rate under the LP and HP treatments were significantly lower than those under the NP treatment. Next, the microstructure difference of tiller buds in the late development stage (S4) at three P levels was analyzed. The number of internode cells and vascular bundles were significantly lower in the LP treatments than in the NP treatments. The relative expression levels of eight P transport genes, eight hormone-related genes and four bud development genes at the tiller bud developmental stage (S2-S4) and the tiller bud re-tillering stage were analyzed with real-time polymerase chain reaction. The results showed that the expression trends for most P transport genes, hormone-related genes and bud development genes from S2 to S4 were diversified at different P levels, and the expression levels were also different at different P levels. In the tiller bud re-tillering stage, the expression levels of seven P transport genes and six hormone-related genes showed a downward trend with increasing P level. REV expression level decreased under LP and HP conditions. TB1 expression level increased under HP condition. Therefore, we conclude that P deficiency inhibits tiller bud development and re-tillering, and that P depends on the expression of REV and TB1 genes and auxin, cytokinin and strigolactones synthesis and transporter genes to mediate tiller bud development and re-tillering.

Comparative analysis of two kinds of garlic seedings: qualities and transcriptional landscape.

BACKGROUND: Facility cultivation is widely applied to meet the increasing demand for high yield and quality, with light intensity and light quality being major limiting factors. However, how changes in the light environment affect development and quality are unclear in garlic. When garlic seedlings are grown, they can also be exposed to blanching culture conditions of darkness or low-light intensity to ameliorate their appearance and modify their bioactive compounds and flavor. RESULTS: In this study, we determined the quality and transcriptomes of 14-day-old garlic and blanched garlic seedlings (green seedlings and blanched seedlings) to explore the mechanisms by which seedlings integrate light signals. The findings revealed that blanched garlic seedlings were taller and heavier in fresh weight compared to green garlic seedlings. In addition, the contents of allicin, cellulose, and soluble sugars were higher in the green seedlings. We also identified 3,872 differentially expressed genes between green and blanched garlic seedlings. The Kyoto Encyclopedia of Genes and Genomes analysis suggested enrichment for plant-pathogen interactions, phytohormone signaling, mitogen-activated protein kinase signaling, and other metabolic processes. In functional annotations, pathways related to the growth and formation of the main compounds included phytohormone signaling, cell wall metabolism, allicin biosynthesis, secondary metabolism and MAPK signaling. Accordingly, we identified multiple types of transcription factor genes involved in plant-pathogen interactions, plant phytohormone signaling, and biosynthesis of secondary metabolites among the differentially expressed genes between green and blanched garlic seedlings. CONCLUSIONS: Blanching culture is one facility cultivation mode that promotes chlorophyll degradation, thus changing the outward appearance of crops, and improves their flavor. The large number of DEGs identified confirmed the difference of the regulatory machinery under two culture system. This study increases our understanding of the regulatory network integrating light and darkness signals in garlic seedlings and provides a useful resource for the genetic manipulation and cultivation of blanched garlic seedlings.

Molecular mechanism of phosphorous signaling inducing anthocyanin accumulation in Arabidopsis.

Anthocyanins, flavonoid compounds derived from secondary metabolic pathways, play important roles in various biological processes. Phosphorus (P) is an essential macroelement for plant growth and development, and P-starvation usually results in anthocyanin accumulation. However, the molecular mechanism of P deficiency promotes anthocyanin biosynthesis has not been well characterized. Here, we provided evidence that the P signaling core protein PHOSPHATE STARVATION RESPONSE1 (PHR1) is physically associate with transcription factors (TFs) involved in anthocyanidin biosynthesis, including PRODUCTION OF ANTHOCYANIN PIGMENTS1 (PAP1/MYB75), MYB DOMAIN PROTEIN 113 (MYB113) and TRANSPARENT TESTA 8 (TT8). PHR1 and its homologies positively regulated anthocyanin accumulation in Arabidopsis seedlings under P-deficient conditions. Disruption of PHR1 simultaneously rendered seedlings hyposensitive to limiting P, whereas the overexpression of PHR1 enhanced P- deficiency-induced anthocyanin accumulation. Genetic analysis demonstrated that 35S:PHR1-2HA-5 seedlings partially recovers the P deficiency insensitive phenotype of myb-RNAi and tt8 mutants. In summary, our study indicated that protein complexes formed by PHR1 and MBW complex directly mediate the process of P-deficiency-induced anthocyanin accumulation, providing a new mechanistic understanding of how P-deficient signaling depends on the endogenous anthocyanin synthesis pathway to promote anthocyanin accumulation in Arabidopsis.

CRISPR/Cas9 Mutant Rice Ospmei12 Involved in Growth, Cell Wall Development, and Response to Phytohormone and Heavy Metal Stress.

Pectin is one of the constituents of the cell wall, distributed in the primary cell wall and middle lamella, affecting the rheological properties and the cell wall stickiness. Pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) are the most important factors for modifying methyl esterification. In this study, 45 PMEI genes from rice (Oryza sativa L.) were screened by bioinformatics tools, and their structure, motifs, cis-acting elements in the promoter region, chromosomal distribution, gene duplication, and phylogenetic relationship were analyzed. Furthermore, CRISPR/Cas9 was used to edit the OsPMEI12 (LOC_Os03G01020) and two mutant pmei12 lines were obtained to explore the functions of OsPMEI in plant growth and development, and under cadmium (Cd) stress. Compared to wild type (WT) Nipponbare, the second inverted internodes of the mutant plants shortened significantly, resulting in the reduction in plant height at mature stage. The seed setting rate, and fresh and dry weights of the mutants were also decreased in mutant plants. In addition, the pectin methylation of pmei12 lines is decreased as expected, and the pectin content of the cell wall increased at both seedling and maturity stages; however, the cellulose and hemicellulose increased only at seedling stage. Interestingly, the growth of the pmei12 lines was better than the WT in both normal conditions and under two phytohormone (GA3 and NAA) treatments at seedling stage. Under Cd stress, the fresh and dry weights were increased in pmei12 lines. These results indicated that OsPMEI12 was involved in the regulation of methyl esterification during growth, affected cell wall composition and agronomic traits, and might play an important role in responses to phytohormones and stress.

Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress.

BACKGROUND: A reasonable supply of nitrogen (N) fertilizer is essential for obtaining high-quality, high-level, and stable potato yields, and an improvement in the N utilization efficiency can effectively reduce N fertilizer use. It is important to use accurate, straightforward, and efficient transgenic breeding techniques for the identification of genes that can improve nitrogen use efficiency, thus enabling us to achieve the ultimate goal of breeding N-efficient potato varieties. In recent years, some of the mechanisms of miRNAs have been elucidated via the analysis of the correlation between the expression levels of potato miRNA target genes and regulated genes under conditions of stress, but the role of miRNAs in the inhibition/expression of key genes regulating N metabolism under N stress is still unclear. Our study aimed to identify the role played by specific enzymes and miRNAs in the responses of plants to N stress. RESULTS: The roots and leaves of the N-efficient potato variety, Yanshu4 ("Y"), and N-inefficient potato variety, Atlantic ("D"), were collected at the seedling and budding stages after they were exposed to different N fertilizer treatments. The miRNAs expressed differentially under the two types of N stress and their corresponding target genes were first predicted using miRNA and degradome analysis. Then, quantitative polymerase chain reaction (qRT-PCR) was performed to verify the expression of differential miRNAs that were closely related to N metabolism. Finally, the shearing relationship between stu-miR396-5p and its target gene StNiR was determined by analyzing luciferase activity levels. The results showed that NiR activity increased significantly with an increase in the applied N levels from the seedling stage to the budding stage, and NiR responded significantly to different N treatments. miRNA sequencing enabled us to predict 48 families with conserved miRNAs that were mainly involved in N metabolism, carbon metabolism, and amino acid biosynthesis. The differences in the expression of the following miRNAs were identified via screening (high expression levels and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, and stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in N metabolism were NiR, NiR1, NRT2.5, and NRT2.7. qRT-PCR analysis showed that there were significant differences in the expression levels of stu-miR396-5p, stu-miR8036-3p, and stu-miR482a-3p in the leaves and roots of the Yanshu4 and Atlantic varieties at the seedling and budding stages under conditions that involved no N and excessive N application; the expression of these miRNAs was induced in response to N stress. The correlation between the differential expression of stu-miR396-5p and its corresponding target gene NiR was further verified by determining the luciferase activity level and was found to be strongly negative. CONCLUSION: The activity of NiR was significantly positively correlated with N application from the seedling to the budding stage. Differential miRNAs and target genes showed a many-to-many relationship with each other. The expression of stu-miR396-5p, stu-miR482a-3p, and stu-miR8036-3p in the roots and leaves of the Yanshu4 and Atlantic varieties at the seedling and budding stages was notably different under two types of N stress. Under two types of N stress, stu-miR396-5p was down-regulated in Yanshu4 in the seedling-stage and shoot-stage roots, and up-regulated in seedling-stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in the seedling and shoot stages. The expression of stu-miR8036-3p was up-regulated in the leaves and roots at the seedling and budding stages, and down-regulated in roots under both types of N stress. The gene expressing the key enzyme involved in N metabolism, StNiR, and the stu-miR396-5p luciferase assay reporter gene had a strong regulatory relationship with each other. This study provides candidate miRNAs related to nitrogen metabolism and highlights that differential miRNAs play a key role in nitrogen stress in potato, providing insights for future research on miRNAs and their target genes in nitrogen metabolic pathways and breeding nitrogen-efficient potatoes.

Acclimation Strategy of Masson Pine (Pinus massoniana) by Limiting Flavonoid and Terpenoid Production under Low Light and Drought.

Low light and drought often limit the growth and performance of Masson pines (Pinus massoniana) in the subtropical forest ecosystem of China. We speculated that stress-induced defensive secondary metabolites, such as flavonoids and terpenoids, might influence the growth of Masson pines, considering the existence of tradeoffs between growth and defense. However, the mechanisms of Masson pines responsive to low light and drought at the levels of these two metabolites remain unclear. In the present work, the compositions of flavonoids and terpenoids, as well as their biosynthetic pathways, were revealed through metabolome and transcriptome analyses, respectively, coupled with a study on carbon allocation using a 13CO2-pulse-labeling experiment in two-year-old seedlings under low light (LL), drought (DR), and their combined stress (DL) compared to a control (CK). A total of 35 flavonoids and derivatives (LL vs. CK: 18; DR vs. CK: 20; and DL vs. CK: 18), as well as 29 terpenoids and derivatives (LL vs. CK: 23; DR vs. CK: 13; and DL vs. CK: 7), were differentially identified in the leaves. Surprisingly, most of them were decreased under all three stress regimes. At the transcriptomic level, most or all of the detected DEGs (differentially expressed genes) involved in the biosynthetic pathways of flavonoids and terpenoids were downregulated in phloem and xylem under stress treatments. This indicated that stress treatments limited the production of flavonoids and terpenoids. The reduction in the 13C allocation to stems might suggest that it is necessary for maintaining the growth of Masson pine seedlings at the whole-plant level by attenuating energetic resources to the biosynthetic pathways of flavonoids and terpenoids when facing the occurrence of adverse environments. Our results provide new insight into understanding the acclimation strategy of Masson pines or other conifers in adverse environments.

Deciphering the iPBS retrotransposons based genetic diversity of nanoparticles induced in Vitro seedlings of industrial hemp (Cannabis sativa L.).

BACKGROUND: The phytochemicals contained in hemp are highly significant and can be modified or altered by employing in vitro elicitors like nanoparticles (NPs). Application of NPs type, concentration, and treatment time regulate the germination, growth, and phytohemicals. METHODS AND RESULTS: In vitro sterilized seeds of cannabis were augmented on Murashige and Skoog (MS) medium supplemented with silver (Ag) and titanium dioxide (TiO2) nanoparticles at different concentrations (0, 200, 400, 800, 1200 and 1600 mg/L) for one month. Results revealed that supplementation of NPs resulted in reduced germination (%), root length and longer shoots and seedling fresh wt compared to control. CONCLUSIONS: Maximum germination was recorded on MS medium supplemented with 1600 mg/L TiO2NPs (92.50%) followed by 1600 mg/L AgNPs (80.00%). Supplementation of 800 mg/L AgNPs yielded longer shoots, roots, seedlings fresh weight, and chlorophyll-b contents compared to all other treatments. Whereas, maximum chlorophyll-a, carotenoids, and MDA contents were attributed to 1200 mg/L TiO2NPs. PCR results using eight iPBS-retrotransposons primers yielded a total of 101 bands with 98 polymorphic bands. Whereas, minimum (0.28) and maximum (0.42) gene diversity was associated with 2095 and 2228 primers.

Genetic manipulation of bermudagrass photosynthetic biosynthesis using Agrobacterium-mediated transformation.

Bermudagrass is one of the most extensively used warm-season grasses. It is widely used in landscaping, stadium construction and soil remediation due to its excellent regeneration, trampling and stress tolerances. However, studies on its regulatory mechanism and variety improvement by genetic engineering are still at a standstill, owing to its genetic variability and intrinsic limits linked with some resistance to Agrobacterium infection. In this study, we established a higher efficient Agrobacterium-mediated transformation via screening for vital embryogenic callus and improving infection efficiency. The superior callus was light yellow, hard granular and compact, determined with a differentiation rate of more than 95%. The optimized infestation courses by gentle shaking, vacuuming and sonicating were used. The infested calluses were co-cultured for 3 days, followed by desiccation treatments for 1 day to get higher infection efficiency. Then the CdHEMA1 gene, essential for chlorophyll biosynthesis, was cloned and transferred into bermudagrass to validate the aforementioned optimization procedures integrally. Molecular-level analyses indicated that the CdHEMA1 gene had successfully integrated and was greatly increased in transgenic seedlings. Results of the photosynthetic capacity assessment showed that CdHEMA1 overexpression may considerably enhance the contents of photosynthetic pigments, OJIP curve and reaction center density (RC/CSo) to absorb (ABS/CSo, ABS/CSM) and capture (TRo/CSo) more light energy, hence improve the performance indices PIABS and PICS compared to the wild type. The successful completion of this project would provide a solid platform for further gene function study and molecular breeding of bermudagrass.

Transcriptome and Metabolome Analyses Revealed the Response Mechanism of Quinoa Seedlings to Different Phosphorus Stresses.

Quinoa (Chenopodium quinoa Willd.) is a dicotyledonous annual herb of Family Amaranthaceae and Subfamily Chenopodiaceae. It has high nutritional and economic value. Phosphorus (P) is an essential plant macronutrient, a component of many biomolecules, and vital to growth, development, and metabolism. We analyzed the transcriptomes and metabolomes of Dianli-1299 and Dianli-71 quinoa seedlings, compared their phenotypes, and elucidated the mechanisms of their responses to the phosphorus treatments. Phenotypes significantly varied with phosphorus level. The plants responded to changes in available phosphorus by modulating metabolites and genes implicated in glycerophospholipid, glycerolipid and glycolysis, and glyconeogenesis metabolism. We detected 1057 metabolites, of which 149 were differentially expressed (DEMs) and common to the control (CK) vs. the low-phosphorus (LP) treatment samples, while two DEMs were common to CK vs. the high-phosphorus (HP) treatment samples. The Kyoto Encyclopedia of genes and genomes (KEGG) annotated 29,232 genes, of which 231 were differentially expressed (DEGs) and common to CK vs. LP, while one was common to CK vs. HP. A total of 15 DEMs and 11 DEGs might account for the observed differences in the responses of the quinoa seedlings to the various phosphorus levels. The foregoing results may provide a theoretical basis for improving the phosphorus utilization efficiency in quinoa.