Genome-wide identification of the Q-type C2H2 zinc finger protein gene family and expression analysis under abiotic stress in lotus (Nelumbo nucifera G.).

BACKGROUND: Lotus (Nelumbo nucifera G.) is an important aquatic plant with high ornamental, economic, cultural and ecological values, but abiotic stresses seriously affect its growth and distribution. Q-type C2H2 zinc finger proteins (ZFPs) play an important role in plant growth development and environmental stress responses. Although the Q-type C2H2 gene family has been identified in some plants, limited reports has been carried out it in lotus. RESULTS: In this study, we identified 45 Q-type NnZFP members in lotus. Based on the phylogenetic tree, these Q-type NnZFP gene family members were divided into 4 groups, including C1-1i, C1-2i, C1-3i and C1-4i. Promoter cis-acting elements analysis indicated that most Q-type NnZFP gene family members in lotus were associated with response to abiotic stresses. Through collinearity analyses, no tandem duplication gene pairs and 14 segmental duplication gene pairs were identified, which showed that duplication events might play a key role in the expansion of the Q-type NnZFP gene family. The synteny results suggested that 54 and 28 Q-type NnZFP genes were orthologous to Arabidopsis and rice, respectively. The expression patterns of these Q-type NnZFP genes revealed that 30 Q-type NnZFP genes were expressed in at least one lotus tissue. Nn5g30550 showed relatively higher expression levels in all tested tissues. 12 genes were randomly selected with at least one gene from each phylogenetic clade, and the expression of these selected genes were confirmed by qRT-PCR (quantitative real-time polymerase chain reaction). The results indicated that Q-type NnZFP genes were extensively involved in cadmium, drought, salt and cold stresses responses. Among them, 11 genes responded to at least three different stress treatments, especially Nn2g12894, which induced by all four treatments. CONCLUSIONS: These results could increase our understanding of the characterization of the Q-type NnZFP gene family and provide relevant information for further functional analysis of Q-type NnZFP genes in plant development, and abiotic stress tolerance in lotus.

Multi-omic dissection of the cold resistance traits of white water lily.

The white water lily (Nymphaea candida), exemplifying nature's resilience, thrives in the high-altitude terrains of Xinjiang, China, serving as an ideal model for investigating cold adaptation mechanisms in aquatic plants. This study meticulously elucidates the complex cold adaptation mechanisms of the white water lily through a comprehensive and integrated methodological approach. We discovered that the water lily undergoes ecodormancy in winter, retaining high cellular viability and growth potential. During overwintering, the white water lily demonstrates effective resource reallocation, a process facilitated by morphological adjustments, thereby strengthening its resistance to cold temperatures. This enhancement is achieved particularly through the compartmentalization of large vacuoles, the accumulation of osmoregulatory substances, and an increased antioxidant capacity. We established the first exhaustive full-length transcriptome for the white water lily. A subsequent comprehensive analysis of the transcriptome, phytohormones, and metabolome uncovered a multifaceted regulatory network orchestrating cold adaptation. Our research spotlights phytohormone signaling, amino acid metabolism, and circadian rhythms as key elements in the water lily's defense against cold. The results emphasize the critical role of nitrogen metabolism, especially amino acid-related pathways, during cold stress. Metabolite profiling revealed the importance of compounds like myo-inositol and L-proline in enhancing cold tolerance. Remarkably, our study demonstrates that the white water lily notably diminishes the utilization of unsaturated fatty acids in its temperature regulation strategies. In conclusion, this research substantially enriches our understanding of the white water lily's intricate cold adaptation mechanisms, offering new perspectives on the adaptive strategies of aquatic plants and potential applications in agricultural advancement.

Purification Effect of Water Eutrophication Using the Mosaic System of Submerged-Emerged Plants and Growth Response.

Aquatic plants play a crucial role in the sustainable management of eutrophic water bodies, serving as a valuable tool for water purification. However, the effectiveness of using aquatic plants for improving water quality is influenced by landscape considerations. In practical applications, challenges arise concerning low purification efficiency and compromised aesthetic appeal when utilizing plants for water purification. To address these issues, this study aimed to examine the impact of aquatic plants on the purification of simulated landscape water bodies, specifically focusing on the effectiveness of the mosaic system of submerged-emerged plants in remediating eutrophic water bodies. Our findings indicated that individual aquatic plants exhibited limited efficacy in pollutant (total nitrogen, total phosphorus, ammonia nitrogen, and chemical oxygen demand) removal. However, when combined in appropriate proportions, submerged plants could enhance species growth and improve the purification efficiency of polluted water bodies. Notably, the mosaic system of submerged-emerged plants neither significantly promoted nor inhibited the growth of each other, but it effectively removed pollutants from the simulated water bodies and inhibited turbidity increase. The comprehensive evaluation ranked the purification capacity as Canna indica-submerged plants combination (C + S) > Thalia dealbata-submerged plants combination (T + S) > Iris pseudacorus-submerged plants combination (I + S) > Lythrum salicaria-submerged plants combination (L + S). Both C + S and T + S configurations effectively mitigated the rise of water turbidity and offered appealing landscape benefits, making them viable options for practical applications in urban landscape water bodies. Our study highlights that a submerged-emerged mosaic combination is a means of water purification that combines landscape aesthetics and purification efficiency.

NnSnRK1-NnATG1-mediated autophagic cell death governs flower bud abortion in shaded lotus.

Many plants can terminate their flowering process in response to unfavourable environments, but the mechanisms underlying this response are poorly understood. In this study, we observed that the lotus flower buds were susceptible to abortion under shaded conditions. The primary cause of abortion was excessive autophagic cell death (ACD) in flower buds. Blockade of autophagic flux in lotus flower buds consistently resulted in low levels of ACD and improved flowering ability under shaded conditions. Further evidence highlights the importance of the NnSnRK1-NnATG1 signalling axis in inducing ACD in lotus flower buds and culminating in their timely abortion. Under shaded conditions, elevated levels of NnSnRK1 activated NnATG1, which subsequently led to the formation of numerous autophagosome structures in lotus flower bud cells. Excessive autophagy levels led to the bulk degradation of cellular material, which triggered ACD and the abortion of flower buds. NnSnRK1 does not act directly on NnATG1. Other components, including TOR (target of rapamycin), PI3K (phosphatidylinositol 3-kinase) and three previously unidentified genes, appeared to be pivotal for the interaction between NnSnRK1 and NnATG1. This study reveals the role of autophagy in regulating the abortion of lotus flower buds, which could improve reproductive success and act as an energy-efficient measure in plants.

Analysis of bZIP gene family in lotus (Nelumbo) and functional study of NnbZIP36 in regulating anthocyanin synthesis.

BACKGROUND: The basic leucine zipper (bZIP) family is a predominant group of transcription factors in plants, involved in regulating plant growth, development, and response to stressors. Additionally, the bZIP gene family has a key role in anthocyanin production. Despite the significant role of bZIP genes in plants, their potential contribution in lotus remains understudied. RESULTS: A total of 124 bZIP genes (59 NnbZIPs and 65 NlbZIPs) were identified from genomes of two lotus species. These genes were classified into 13 groups according to the grouping principle of the Arabidopsis bZIP gene family. Analysis of promoter cis-acting elements indicated that most bZIP gene family members in lotus are associated with response to abiotic stresses. The promoters of some bZIP genes contain MYB binding sites that regulate anthocyanin synthesis. We examined the anthocyanin content of the petals from three different colored lotus, combined with transcriptome data analysis and qRT-PCR results, showing that the expression trends of NnbZIP36 and the homologous gene NlbZIP38 were significantly correlated with the anthocyanin content in lotus petals. Furthermore, we found that overexpression of NnbZIP36 in Arabidopsis promoted anthocyanin accumulation by upregulating the expression of genes (4CL, CHI, CHS, F3H, F3'H, DFR, ANS and UF3GT) related to anthocyanin synthesis. CONCLUSIONS: Our study enhances the understanding of the bZIP gene family in lotus and provides evidence for the role of NnbZIP36 in regulating anthocyanin synthesis. This study also sets the stage for future investigations into the mechanism by which the bZIP gene family regulates anthocyanin biosynthesis in lotus.

Screening of Early Flowering Lotus (Nelumbo nucifera Gaertn.) Cultivars and Effects of Different Cultivars on Flowering Period.

Flowering time is an important trait that determines the breeding process of ornamental plants. The flowering period of lotus (Nelumbo nucifera Gaertn.) is mainly concentrated in June-August. During this period, the weather is hot and there are few tourists, which made many lotus scenic spots difficult to operate. People have a strong demand for early flowering lotus cultivars. In this paper, 30 lotus cultivars with high ornamental value were selected as materials and their phenological periods were observed for two consecutive years in 2019 and 2020. A number of cultivars with early flowering potential and stable flowering periods, such as 'Fenyanzi', 'Chengshanqiuyue', 'Xianghumingyue' and 'Wuzhilian', were screened by K-Means clustering method. The relationship between accumulated temperature and flowering time of 19 lotus cultivars at different growth stages was analyzed. It was found that lotus cultivars with early flowering traits could adapt well to the changes of early environmental temperature and were not affected by low temperature. On the other hand, by analyzing the relationship between different traits and flowering time of three typical cultivars, such as rhizome weight, phenological period, etc., it shows that the nutrient content of the rhizome and the early morphology of plants will affect the flowering time. These results provide a reference for the formation of a systematic lotus early flowering cultivar breeding mechanism and the establishment of a perfect flowering regulation technology system, which can further improve the ornamental value of lotus and promote industrial development.

Improving the efficacy of different life-form macrophytes in phytoremediation of artificial eutrophic water by combined planting.

Phytoremediation of the eutrophic water bodies by using various macrophytes has long been considered effective and economical. However, the understanding of combining macrophytes to maximize efficacy in the restoration is still limited. In this study, three different life-form macrophytes were employed to explore the optimal plant combination of eutrophic water purification, including Pontederia cordata L. (E: emergent), Pistia stratiotes L. (F: floating), and Hydrilla verticillata (L. f.) Royle (S: submerged). The effects on water quality, removal of the excess nutrients (TN, NH3-N, NO3-N, and TP) in the water, along with the growth response and the nutrient accumulation of the macrophytes were investigated both individually and in combination. The phytoremediation of every single macrophyte was significantly improved by combined planting and increasing the diversity of the combination led to better enhancements. In general, the treatment with macrophytes in three life forms (EFS) not only resulted in the highest removal rates of the TN, NH3-N, NO3-N, and TP (40.89, 33.50, 46.81, and 43.55%, respectively) but also decreased the turbidity and increased the dissolved oxygen more effectively and efficiently. Furthermore, EFS mitigated the environmental stress of plants and promoted the accumulation of TN and TP in them, especially the emergent macrophyte P. cordata. The combinations with macrophyte in two life forms (EF, ES, and FS) also exhibited unique strengths: the removal efficacy of TN (39.25%) and TP (46.16%) in FS, and NO3-N in EF (48.54%) and ES (49.90%) were also at the forefront; the biomass and nutrient content of the submerged macrophyte H. verticillata in ES were the highest. Moreover, a strong correlation between the eutrophic factors and the plant physiological indexes was observed. These findings highlighted the role of combined planting in phytoremediation and provided a valuable reference for the development of ecological restoration for eutrophic ecosystems.

Comparative transcriptome analysis reveals the regulatory mechanisms of two tropical water lilies in response to cold stress.

BACKGROUND: Tropical water lily is an aquatic plant with high ornamental value, but it cannot overwinter naturally at high latitudes. The temperature drop has become a key factor restricting the development and promotion of the industry. RESULTS: The responses of Nymphaea lotus and Nymphaea rubra to cold stress were analyzed from the perspective of physiology and transcriptomics. Under the cold stress, Nymphaea rubra had obvious leaf edge curling and chlorosis. The degree of peroxidation of its membrane was higher than that of Nymphaea lotus, and the content of photosynthetic pigments also decreased more than that of Nymphaea lotus. The soluble sugar content, SOD enzyme activity and CAT enzyme activity of Nymphaea lotus were higher than those of Nymphaea rubra. This indicated that there were significant differences in the cold sensitivity of the two varieties. GO enrichment and KEGG pathway analysis showed that many stress response genes and pathways were affected and enriched to varying degrees under the cold stress, especially plant hormone signal transduction, metabolic pathways and some transcription factor genes were from ZAT gene family or WKRY gene family. The key transcription factor ZAT12 protein in the cold stress response process has a C2H2 conserved domain, and the protein is localized in the nucleus. Under the cold stress, overexpression of the NlZAT12 gene in Arabidopsis thaliana increased the expression of some cold-responsive protein genes. The content of reactive oxygen species and MDA in transgenic Arabidopsis thaliana was lower, and the content of soluble sugar was higher, indicating that overexpression of NlZAT12 can improve the cold tolerance of Arabidopsis thaliana. CONCLUSION: We demonstrate that ethylene signalling and reactive oxygen species signalling play critical roles in the response of the two cultivars to cold stress. The key gene NlZAT12 for improving cold tolerance was identified. Our study provides a theoretical basis for revealing the molecular mechanism of tropical water lily in response to cold stress.

Cytological and Molecular Mechanism of Low Pollen Grain Viability in a Germplasm Line of Double Lotus.

Self-fertilization rate is an essential index of lotus reproductive system development, and pollen activity is a key factor affecting lotus seed setting rate. Based on cytology and molecular biology, this study addresses the main reasons for the low self-set rate of double lotus. It takes two different double lotus breeds into consideration, namely 'Sijingganshan' with a low self-crossing rate and 'Jinfurong' with a high self-crossing rate. Cytological analysis results showed that the pollen abortion caused by excessive degradation of tapetum during the single phase was the root cause for the low self-mating rate of double lotus. Subsequent transcriptome analysis revealed that the gene NnPTC1 related to programmed tapetum cell death was significantly differentially expressed during the critical period of abortion, which further verified the specific expression of NnPTC1 in anthers. It was found that the expression level of NnPTC1 in 'Sijingganshan' at the mononuclear stage of its microspore development was significantly higher than that of 'Jinfurong' at the same stage. The overexpression of NnPTC1 resulted in the premature degradation of the tapetum and significantly decreased seed setting rate. These results indicated that the NnPTC1 gene regulated the pollen abortion of double lotus. The mechanism causing a low seed setting rate for double lotus was preliminarily revealed, which provided a theoretical basis for cultivating lotus varieties with both flower and seed.

Integrated Transcriptome and Targeted Metabolite Analysis Reveal miRNA-mRNA Networks in Low-Light-Induced Lotus Flower Bud Abortion.

Most Nelumbo nucifera (lotus) flower buds were aborted during the growing season, notably in low-light environments. How lotus produces so many aborted flower buds is largely unknown. An integrated transcriptome and targeted metabolite analysis was performed to reveal the genetic regulatory networks underlying lotus flower bud abortion. A total of 233 miRNAs and 25,351 genes were identified in lotus flower buds, including 68 novel miRNAs and 1108 novel genes. Further enrichment analysis indicated that sugar signaling plays a potential central role in regulating lotus flower bud abortion. Targeted metabolite analysis showed that trehalose levels declined the most in the aborting flower buds. A potential regulatory network centered on miR156 governs lotus flower bud abortion, involving multiple miRNA-mRNA pairs related to cell integrity, cell proliferation and expansion, and DNA repair. Genetic analysis showed that miRNA156-5p-overexpressing lotus showed aggravated flower bud abortion phenotypes. Trehalose-6-P synthase 1 (TPS1), which is required for trehalose synthase, had a negative regulatory effect on miR156 expression. TPS1-overexpression lotus showed significantly decreased flower bud abortion rates both in normal-light and low-light environments. Our study establishes a possible genetic basis for how lotus produces so many aborted flower buds, facilitating genetic improvement of lotus' shade tolerance.

Differential efficacy of water lily cultivars in phytoremediation of eutrophic water contaminated with phosphorus and nitrogen.

Excessive inputs of phosphorus (P) and nitrogen (N) trigger eutrophication of the water bodies, which promotes the undesirable growth of algal bloom and deterioration of the water quality, and aquatic biodiversity. Macrophytes provide an environmentally benign and economically viable paradigm for the ecological restoration of eutrophic aquatic ecosystems. Water lily (Nymphaea) is largely used as ornamental plant for landscaping, and it has been documented that water lily possesses the potentiality in mitigating polluted aquatic environments. In the present study, water lily cultivars Nymphaea Texas Dawn (NTD), Nymphaea Colorado (NC), Nymphaea Madame Wilfron Gonnère (NMWG), and Nymphaea Sunshine Princess (NSP) were investigated for their potency in alleviating the eutrophication. The concentrations of total P and total N were significantly higher in the leaves of NC and NSP compared with NTD and NMWG. Therefore, NC and NSP were selected for subsequent studies to decipher their recuperation efficacy on eutrophic waters at different growth stages. NC and NSP significantly reduced the concentrations of eutrophication indicators i.e., total P, NH4+-N, and chemical oxygen demand in different gradients of the simulated eutrophic water in a growth-dependent manner. On the contrary, NC and NSP triggered a significant increase in the concentration of dissolved oxygen particularly at the seedling stage. Notably, the concentrations of total P (shoot and root) and total N (root) were relatively higher in NSP than NC. The study thus revealed a growth-dependent differential efficacy of NSP and NC in mitigating the different eutrophic waters.

Regulation of Flowering Timing by ABA-NnSnRK1 Signaling Pathway in Lotus.

The lotus produces flower buds at each node, yet most of them are aborted because of unfavorable environmental changes and the mechanism remains unclear. In this work, we proposed a potential novel pathway for ABA-mediated flower timing control in the lotus, which was explored by combining molecular, genetic, transcriptomic, biochemical, and pharmacologic approaches. We found that the aborting flower buds experienced extensive programmed cell death (PCD). The hormonal changes between the normal and aborting flower buds were dominated by abscisic acid (ABA). Seedlings treated with increasing concentrations of ABA exhibited a differential alleviating effect on flower bud abortion, with a maximal response at 80 µM. Transcriptome analysis further confirmed the changes of ABA content and the occurrence of PCD, and indicated the importance of PCD-related SNF1-related protein kinase 1 (NnSnRK1). The NnSnRK1-silenced lotus seedlings showed stronger flowering ability, with their flower:leaf ratio increased by 40%. When seedlings were treated with ABA, the expression level and protein kinase activity of NnSnRK1 significantly decreased. The phenotype of NnSnRK1-silenced seedlings could also be enhanced by ABA treatment and reversed by tungstate treatment. These results suggested that the decline of ABA content in lotus flower buds released its repression of NnSnRK1, which then initiated flower bud abortion.

Effects of ethylene biosynthesis and signaling on oxidative stress and antioxidant defense system in Nelumbo nucifera G. under cadmium exposure.

Water contamination with cadmium (Cd) is a global environmental problem and its remediation becomes urgent. Phytoremediation using ornamental plants has attracted much attention for its advantages of cost-effectiveness and beautification of the environment. Nelumbo nucifera G. is a popular ornamental aquatic macrophyte with fast growth, large biomass, and high capacities for Cd accumulation and removal. However, information about Cd resistance and defense responses in N. nucifera is rather scarce, which restricts its large-scale utilization for phytoremediation. The phytohormone ethylene plays an important role in plant resistance to Cd stress, but the underlying mechanism remains unclear. In this study, we investigated morphophysiological responses of N. nucifera seedlings to Cd stress, and focused on the effects of ethylene on oxidative damage, Cd accumulation, and antioxidant defense system at the metabolic and transcript levels in leaves under Cd stress. Our results showed that Cd exposure led to leaf chlorosis and necrosis, coupled with an increase in contents of hydrogen peroxide, electrolyte leakage, and malondialdehyde, and decrease in chlorophyll content. Exogenous ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) application intensified Cd-induced stress responses and Cd accumulation, and increased ethylene production by inducing ACC synthase (ACS) gene NnACS. Such enhanced ethylene emission inhibited catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities, and modulated ascorbate (AsA) and glutathione (GSH) accumulation through transcriptional regulation of their respective metabolic genes. After ethylene action inhibitor silver thiosulfate (STS) supplementation, Cd-induced oxidative damage was abolished, and Cd content declined but still at a relatively high level. Blocking of ethylene perception by STS inhibited ethylene biosynthesis; enhanced CAT, APX, and GR activities and their transcript levels; increased AsA accumulation via inducing its biosynthetic genes; but reduced GSH content and NnGSH2 expression level. These results suggest that ethylene biosynthesis and signaling play an important role in N. nucifera response to Cd stress, and maintaining appropriate ethylene level and low ethylene sensitivity could improve its Cd tolerance via efficient antioxidant defenses.

Genome-wide transcriptional analysis of submerged lotus reveals cooperative regulation and gene responses.

Flooding severely limits plant growth even for some aquatic plants. Although much work has been done on submergence response of some important crop plants, little is known about the response mechanism of aquatic plants, i.e. lotus (Nelumbo nucifera). In this study, we investigated the genome-wide regulation lotus genes in response to submergence stress by high-throughput mRNA sequencing. A total of 4002 differentially expressed genes (DEGs) in lotus upon submergence stress. Among them, 1976 genes were up-regulated and 2026 down-regulated. Functional annotation of these genes by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis revealed that they were mainly involved in processes of oxidation-reduction, abiotic stimuli, cellular metabolism and small molecule metabolism. Based on these data, previous work and quantitative RT-PCR (RT-qPCR) validation, we constructed a cooperative regulation network involved in several important DEGs in regards to the antioxidant system, disease resistance, hypoxia resistance and morphological adaptation. Further work confirmed that several innate immunity genes were induced during submergence and might confer higher resistance to lotus rot disease. In conclusion, these results provide useful information on molecular mechanisms underlying lotus responses to submergence stress.

Genome-wide analysis of the Solanum tuberosum (potato) trehalose-6-phosphate synthase (TPS) gene family: evolution and differential expression during development and stress.

BACKGROUND: Trehalose-6-phosphate synthase (TPS) serves important functions in plant desiccation tolerance and response to environmental stimuli. At present, a comprehensive analysis, i.e. functional classification, molecular evolution, and expression patterns of this gene family are still lacking in Solanum tuberosum (potato). RESULTS: In this study, a comprehensive analysis of the TPS gene family was conducted in potato. A total of eight putative potato TPS genes (StTPSs) were identified by searching the latest potato genome sequence. The amino acid identity among eight StTPSs varied from 59.91 to 89.54%. Analysis of dN/dS ratios suggested that regions in the TPP (trehalose-6-phosphate phosphatase) domains evolved faster than the TPS domains. Although the sequence of the eight StTPSs showed high similarity (2571-2796 bp), their gene length is highly differentiated (3189-8406 bp). Many of the regulatory elements possibly related to phytohormones, abiotic stress and development were identified in different TPS genes. Based on the phylogenetic tree constructed using TPS genes of potato, and four other Solanaceae plants, TPS genes could be categorized into 6 distinct groups. Analysis revealed that purifying selection most likely played a major role during the evolution of this family. Amino acid changes detected in specific branches of the phylogenetic tree suggests relaxed constraints might have contributed to functional divergence among groups. Moreover, StTPSs were found to exhibit tissue and treatment specific expression patterns upon analysis of transcriptome data, and performing qRT-PCR. CONCLUSIONS: This study provides a reference for genome-wide identification of the potato TPS gene family and sets a framework for further functional studies of this important gene family in development and stress response.

Hydrogen Gas Is Involved in Auxin-Induced Lateral Root Formation by Modulating Nitric Oxide Synthesis.

Metabolism of molecular hydrogen (H2) in bacteria and algae has been widely studied, and it has attracted increasing attention in the context of animals and plants. However, the role of endogenous H2 in lateral root (LR) formation is still unclear. Here, our results showed that H2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H2 production in tomato seedlings, and a contrasting response was observed in the presence of N-1-naphthyphthalamic acid (NPA), an auxin transport inhibitor. NPA-triggered the inhibition of H2 production and thereafter lateral root development was rescued by exogenously applied H2. Detection of endogenous nitric oxide (NO) by the specific probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in both NAA- and H2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H2 were prevented by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO) and the inhibitor of nitrate reductase (NR; an important NO synthetic enzyme). Molecular evidence confirmed that some representative NO-targeted cell cycle regulatory genes were also induced by H2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H2, Arabidopsis mutants nia2 (in particular) and nia1 (two nitrate reductases (NR)-defective mutants) exhibited defects in lateral root length. Together, these results demonstrated that auxin-induced H2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis.

Identification of Submergence-Responsive MicroRNAs and Their Targets Reveals Complex MiRNA-Mediated Regulatory Networks in Lotus (Nelumbo nucifera Gaertn).

MicroRNAs (miRNAs) are endogenous non-coding RNAs with important regulatory functions in plant development and stress responses. However, their population abundance in lotus (Nelumbo nucifera Gaertn) has so far been poorly described, particularly in response to stresses. In this work, submergence-related miRNAs and their target genes were systematically identified, compared, and validated at the transcriptome-wide level using high-throughput sequencing data of small RNA, Mrna, and the degradome. A total of 128 known and 20 novel miRNAs were differentially expressed upon submergence. We identified 629 target transcripts for these submergence-responsive miRNAs. Based on the miRNA expression profiles and GO and KEGG annotation of miRNA target genes, we suggest possible molecular responses and physiological changes of lotus in response to submergence. Several metabolic, physiological and morphological adaptations-related miRNAs, i.e., NNU_far-miR159, NNU_gma-miR393h, and NNU_aly-miR319c-3p, were found to play important regulatory roles in lotus response to submergence. This work will contribute to a better understanding of miRNA-regulated adaption responses of lotus to submergence stress.

Genome-Wide Identification and Evolution Analysis of Trehalose-6-Phosphate Synthase Gene Family in Nelumbo nucifera.

Trehalose-6-phosphate synthase (TPS) plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus) genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS). It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The dN /dS ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean, and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II), where each subfamily could be further divided into 2 (I) and 5 (II) subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus.

De novo transcriptome sequencing and discovery of genes related to copper tolerance in Paeonia ostii.

Paeonia ostii, an important ornamental and medicinal plant, grows normally on copper (Cu) mines with widespread Cu contamination of soils, and it has the ability to lower Cu contents in the Cu-contaminated soils. However, very little molecular information concerned with Cu resistance of P. ostii is available. In this study, high-throughput de novo transcriptome sequencing was carried out for P. ostii with and without Cu treatment using Illumina HiSeq 2000 platform. A total of 77,704 All-unigenes were obtained with a mean length of 710 bp. Of these unigenes, 47,461 were annotated with public databases based on sequence similarities. Comparative transcript profiling allowed the discovery of 4324 differentially expressed genes (DEGs), with 2207 up-regulated and 2117 down-regulated unigenes in Cu-treated library as compared to the control counterpart. Based on these DEGs, Gene Ontology (GO) enrichment analysis indicated Cu stress-relevant terms, such as 'membrane' and 'antioxidant activity'. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis uncovered some important pathways, including 'biosynthesis of secondary metabolites' and 'metabolic pathways'. In addition, expression patterns of 12 selected DEGs derived from quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with their transcript abundance changes obtained by transcriptomic analyses, suggesting that all the 12 genes were authentically involved in Cu tolerance in P. ostii. This is the first report to identify genes related to Cu stress responses in P. ostii, which could offer valuable information on the molecular mechanisms of Cu resistance, and provide a basis for further genomics research on this and related ornamental species for phytoremediation.

Transcriptome-Wide Identification of miRNAs and Their Targets from Typha angustifolia by RNA-Seq and Their Response to Cadmium Stress.

MicroRNAs (miRNAs) play important roles in plant responses to environmental stress. In this work, we used high-throughput sequencing to analyze transcriptome and small RNAs (sRNAs) in Typha angustifolia under cadmium (Cd) stress. 57,608,230 raw reads were obtained from deep sequencing of a pooled cDNA library. Sequence assembly and analysis yielded 102,473 unigenes. We subsequently sequenced two sRNA libraries from T. angustifolia with or without Cd exposure respectively. Based on transcriptome data of T. angustifolia, we catalogued and analyzed the sRNAs, resulting in the identification of 114 conserved miRNAs and 41 novel candidate miRNAs in both small RNA libraries. In silico analysis revealed 764 targets for 89 conserved miRNAs and 21 novel miRNAs. Statistical analysis on sequencing reads abundance and experimental validation revealed that 4 conserved and 6 novel miRNAs showed specific expression. Combined with function of target genes, these results suggested that miRNAs might play a role in plant Cd stress response. This study provided the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in T. angustifolia, which provide a framework for further analysis of miRNAs and their role in regulating plant responses to Cd stress.