Exploring domestication pattern in lotus: insights from dispensable genome assembly.

Lotus (Nelumbo nucifera Gaertn.), an important aquatic plant in horticulture and ecosystems, has been cultivated for more than 7000 years and domesticated into three different subgroups: flower lotus, rhizome lotus, and seed lotus. To explore the domesticated regions of each subgroup, re-sequencing data of 371 lotus accessions collected from the public database were aligned to the genome of 'China-Antique (CA)'. Unmapped reads were used to build the dispensable genome of each subgroup using a metagenome-like assembly strategy. More than 27 Mb of the dispensable genome in these three subgroups and the wild group was assembled, of which 11,761 genes were annotated. Some of the contigs in the dispensable genome were similar to the genomic segments of other lotus accessions other than 'CA'. The annotated genes in each subgroup played essential roles in specific developmental processes. Dissection of selective signals in three cultivated subgroups also demonstrated that subgroup-specific metabolic pathways, such as the brassinosteroids metabolism enrichment in FL, associated with these selected genes in each subgroup and the contigs in dispensable genome nearly located in the domesticated regions of each subgroup, respectively. Our data presented a valuable resource for facilitating lotus genomic studies, complemented the helpful information to the reference genome, and shed light on the selective signals of domesticated subgroups.

Proteomic and metabolomic analyses showing the differentially accumulation of NnUFGT2 is involved in the petal red-white bicolor pigmentation in lotus (Nelumbo nucifera).

Bicolor flower lotus is rare with high ornamental value. During the long history of breeding and artificial selection, a very famous lotus cultivar 'Da Sajin' with red and white picotee bicolor petals were obtained. In order to reveal the mechanism underlying the formation of its picotee bicolor pattern in the petal, an integrative metabolomics and proteomics analyses were conducted between red and white parts of its petals. The results showed that the defect of anthocyanidin 3-O-glucosyltransferases (UFGTs) accumulation resulted in the failure of the glycosylation of anthocyanidin, the last step of anthocyanin biosynthesis in white part of the petals. And proteomic data and biochemical analysis showed that the defect of UFGTs accumulation is not related to their transcription, but because of their degradation. Function of one differentially accumulated NnUFGT were proven being involved in anthocyanin biosynthesis through both in-vitro enzyme assay and in-vivo transgenic analyses. This regulation on the protein accumulation of structural genes in anthocyanin biosynthesis was not explored in any other plants, and hence supposed to be a novel mechanism for the formation of picotee bicolor pattern flower. The results not only provide some new insights into the understanding of lotus flower coloration, but also might assist the breeding of flower lotus.

Comparative proteomic analyses of Tartary buckwheat (Fagopyrum tataricum) seeds at three stages of development.

Tartary buckwheat is among the valuable crops, utilized as both food and Chinese herbal medicine. To uncover the accumulation dynamics of the main nutrients and their regulatory mechanism of Tartary buckwheat seeds, microscopic observations and nutrient analysis were conducted which suggested that starch, proteins as well as flavonoid gradually accumulated among seed development. Comparative proteomic analysis of rice Tartary buckwheat at three different developmental stages was performed. A total of 78 protein spots showed differential expression with 74 of them being successfully identified by MALDI-TOF/TOF MS. Among them, granule bound starch synthase (GBSS1) might be the critical enzyme that determines starch biosynthesis, while 11 S seed storage protein and vicilin seemed to be the main globulin and affect seed storage protein accumulation in Tartary buckwheat seeds. Two enzymes, flavanone 3-hydroxylase (F3H) and anthocyanidin reductase (ANR), involved in the flavonoid biosynthesis pathway were identified. Further analysis on the expression profiles of flavonoid biosynthetic genes revealed that F3H might be the key enzyme that promote flavonoid accumulation. This study provides insights into the mechanism of nutrition accumulation at the protein level in Tartary buckwheat seeds and may facilitate in the breeding and enhancement of Tartary buckwheat germplasm.

A stress-associated protein OsSAP8 modulates gibberellic acid biosynthesis by reducing the promotive effect of transcription factor OsbZIP58 on OsKO2.

Gibberellic acid (GA) is a vital phytohormone for plant growth and development. GA biosynthesis is a complex pathway regulated by various transcription factors. Here we report a stress-associated protein 8 (OsSAP8), negatively involved in GA biosynthesis. Overexpression of OsSAP8 in rice resulted in a semi-dwarfism phenotype and reduced endogenous GA3 content. In contrast, an OsSAP8 knockout mutant exhibited higher endogenous GA3 content and slightly increased plant height. Sub-cellular localization analysis of OsSAP8 showed that it could enter the nucleus. Based on electrophoretic mobility shift assay and yeast one hybrid experiments, OsSAP8 was found to bind to the cis-acting regulatory element GADOWNAT of ent-kaurene oxidases (KO2, KO3, KO5). The results from dual-luciferase reporter assays showed that OsSAP8 does not activate LUC reporter gene expression. However, it could interact with basic leucine zipper 58 (OsbZIP58), which has strong transcriptional activation potential on OsKO2. Moreover, the interaction between OsSAP8, rice lesion simulating disease 1-like 1 (OsLOL1), and OsbZIP58 could reduce the promotive effect of transcription factor OsbZIP58 on OsKO2. These results provide some new insights on the regulation of GA biosynthesis in rice.

Comparative transcriptomic analysis provides insight into carpel petaloidy in lotus (Nelumbo nucifera).

Lotus (Nelumbo nucifera) is a highly recognized flower with high ornamental value. Flower color and flower morphology are two main factors for flower lotus breeding. Petaloidy is a universal phenomenon in lotus flowers. However, the genetic regulation of floral organ petaloidy in lotus remains elusive. In this study, the transcriptomic analysis was performed among three organs, including petal, carpel petaloidy, and carpel in lotus. A total of 1,568 DEGs related to carpel petaloidy were identified. Our study identified one floral homeotic gene encoded by the MADS-box transcription factor, AGAMOUS (AG) as the candidate gene for petaloid in lotus. Meanwhile, a predicted labile boundary in floral organs of N. nucifera was hypothesized. In summary, our results explored the candidate genes related to carpel petaloidy, setting a theoretical basis for the molecular regulation of petaloid phenotype.

Metabolomic Analysis on the Petal of 'Chen Xi' Rose with Light-Induced Color Changes.

Flower color is one of the most prominent traits of rose flowers and determines their ornamental value. The color of the "Chen Xi" rose can change from yellow to red during flower blooming. In the present study, the flavonoid metabolites were investigated by the UPLC-ESI-MS/MS from the petals of four successive flower development stages under natural conditions. In total, 176 flavonoid components, including 49 flavones, 59 flavonols, 12 flavanones, 3 isoflavones, 12 anthocyanins, and 41 proanthocyanidins were identified, with some of them being detected for the first time in this study. Additionally, there were 56 compounds that showed differences among comparison groups, mainly being enriched in pathways of isoflavone, flavonoid, flavone, flavonol, phenylpropanoids, and anthocyanin. Among them, it is anthocyanins that allow the rose flower to turn red when exposed to sunlight. To verify this result, compounds from rose petal with shading treatment (S2D) was also detected but could be clearly separated from the four samples under light by clustering and principal component analyses (PCA). Consistent with low anthocyanins accumulation, the flower with shading could not turn red. Moreover, it provides a foundation for further research on the light-induced color modification of flower.

Protein Phosphorylation Response to Abiotic Stress in Plants.

Plants are an important part of nature because as photoautotrophs, they provide a nutrient source for many other living organisms. Due to their sessile nature, to overcome both biotic and abiotic stresses, plants have developed intricate mechanisms for perception of and reaction to these stresses, both on an external level (perception) and on an internal level (reaction). Specific proteins found within cells play crucial roles in stress mitigation by enhancing cellular processes that facilitate the plants survival during the unfavorable conditions. Well before plants are able to synthesize nascent proteins in response to stress, proteins which already exist in the cell can be subjected to an array of posttranslation modifications (PTMs) that permit a rapid response. These activated proteins can, in turn, aid in further stress responses. Different PTMs have different functions in growth and development of plants. Protein phosphorylation, a reversible form of modification has been well elucidated, and its role in signaling cascades is well documented. In this mini-review, we discuss the integration of protein phosphorylation with other components of abiotic stress-responsive pathways including phytohormones and ion homeostasis. Overall, this review demonstrates the high interconnectivity of the stress response system in plants and how readily plants are able to toggle between various signaling pathways in order to survive harsh conditions. Most notably, fluctuations of the cytosolic calcium levels seem to be a linking component of the various signaling pathways.

Genome-wide analysis of AP2/ERF superfamily in lotus (Nelumbo nucifera) and the association between NnADAP and rhizome morphology.

BACKGROUND: The AP2/ERF family is widely present in plants and plays a crucial regulatory role in plant growth and development. As an essential aquatic horticultural model plant, lotus has an increasingly prominent economic and research value. RESULTS: We have identified and analysed the AP2/ERF gene family in the lotus. Initially, 121 AP2/ERF family genes were identified. By analysing their gene distribution and protein structure, and their expression patterns during the development of lotus rhizome, combined with previous studies, we obtained an SNP (megascaffold_20:3578539) associated with lotus rhizome phenotype. This SNP was in the NnADAP gene of the AP2 subfamily, and the changes in SNP (C/T) caused amino acid conversion (proline/leucine). We constructed a population of 95 lotus varieties for SNP verification. Through population typing experiments, we found that the group with SNP CC had significantly larger lotus rhizome and higher soluble sugar content among the population. CONCLUSIONS: In conclusion, we speculate that the alteration of the SNP in the NnADAP can affect the size and sugar content of the lotus rhizome.

A CONSTANS-LIKE gene of Nelumbo nucifera could promote potato tuberization.

MAIN CONCLUSION: CONSTANS-LIKE 5 of Nelumbo nucifera is capable of promoting potato tuberization through CONSTANS-FLOWERING LOCUS T and gibberellin signaling pathways with a probable association with lotus rhizome enlargement. Lotus (Nelumbo nucifera) is an aquatic plant that is affiliated to the Nelumbonaceace family. It is widely used as an ornamental, vegetable, and medicinal herb with its rhizome being a popular vegetable. To explore the molecular mechanism underlying its rhizome enlargement, we conducted a systematic analysis on the CONSTANS-LIKE (COL) gene family, with the results, indicating that this gene plays a role in regulating potato tuber expansion. These analyses included phylogenetic relationships, gene structure, and expressional patterns of lotus COL family genes. Based on these analyses, NnCOL5 was selected for further study on its potential function in lotus rhizome formation. NnCOL5 was shown to be located in the nucleus, and its expression was positively associated with the enlargement of lotus rhizome. Besides, the overexpression of NnCOL5 in potato led to increased tuber weight and starch content under short-day conditions without changing the number of tubers. Further analysis suggested that the observed tuber changes might be mediated by affecting the expression of genes in CO-FT and GA signaling pathways. These results provide valuable insight in understanding the functions of COL gene as well as the enlargement of lotus rhizome.

Metabolomics Analyses of Cotyledon and Plumule Showing the Potential Domestic Selection in Lotus Breeding.

Lotus (Nelumbo nucifera) seeds are widely consumed as functional food or herbal medicine, of which cotyledon (CL) is the main edible part, and lotus plumule (LP) is commonly utilized in traditional Chinese medicine. However, few studies have been conducted to investigate the chemical components of CL and LP in dry lotus seeds, not to mention the comparison between wild and domesticated varieties. In this study, a widely targeted metabolomics approach based on Ultra Performance Liquid Chromatography-electrospray ionization-Tandem mass spectrometry (UPLC-ESI-MS/MS) was utilized to analyze the metabolites in CL and LP of China Antique ("CA", a wild variety) and Jianxuan-17 ("JX", a popular cultivar). A total of 402 metabolites were identified, which included flavonoids (23.08% to 27.84%), amino acids and derivatives (14.18-16.57%), phenolic acids (11.49-12.63%), and lipids (9.14-10.95%). These metabolites were classified into ten clusters based on their organ or cultivar-specific characters. Most of these metabolites were more abundant in LP than in CL for both varieties, except for metabolites belonging to organic acids and lipids. The analysis of differentially accumulated metabolites (DAMs) demonstrated that more than 25% of metabolites detected in our study were DAMs in CL and LP comparing "JX" with "CA", most of which were less abundant in "JX", including 35 flavonoids in LP, 23 amino acids and derivatives in CL, 7 alkaloids in CL, and 10 nucleotides and derivatives in LP, whereas all of 11 differentially accumulated lipids in LP were more abundant in "JX". Together with the fact that the seed yield of "JX" is much higher than that of "CA", these results indicated that abundant metabolites, especially the functional secondary metabolites (mainly flavonoids and alkaloids), were lost during the process of breeding selection.

Advances on Plant Ubiquitylome-From Mechanism to Application.

Post-translational modifications (PTMs) of proteins enable modulation of their structure, function, localization and turnover. To date, over 660 PTMs have been reported, among which, reversible PTMs are regarded as the key players in cellular signaling. Signaling mediated by PTMs is faster than re-initiation of gene expression, which may result in a faster response that is particularly crucial for plants due to their sessile nature. Ubiquitylation has been widely reported to be involved in many aspects of plant growth and development and it is largely determined by its target protein. It is therefore of high interest to explore new ubiquitylated proteins/sites to obtain new insights into its mechanism and functions. In the last decades, extensive protein profiling of ubiquitylation has been achieved in different plants due to the advancement in ubiquitylated proteins (or peptides) affinity and mass spectrometry techniques. This obtained information on a large number of ubiquitylated proteins/sites helps crack the mechanism of ubiquitylation in plants. In this review, we have summarized the latest advances in protein ubiquitylation to gain comprehensive and updated knowledge in this field. Besides, the current and future challenges and barriers are also reviewed and discussed.

Genome-wide identification of MADS-box gene family in sacred lotus (Nelumbo nucifera) identifies a SEPALLATA homolog gene involved in floral development.

BACKGROUND: Sacred lotus (Nelumbo nucifera) is a vital perennial aquatic ornamental plant. Its flower shape determines the horticultural and ornamental values. However, the mechanisms underlying lotus flower development are still elusive. MADS-box transcription factors are crucial in various features of plant development, especially in floral organogenesis and specification. It is still unknown how the MADS-box transcription factors regulate the floral organogenesis in lotus. RESULTS: To obtain a comprehensive insight into the functions of MADS-box genes in sacred lotus flower development, we systematically characterized members of this gene family based on the available genome information. A total of 44 MADS-box genes were identified, of which 16 type I and 28 type II genes were categorized based on the phylogenetic analysis. Furthermore, the structure of MADS-box genes and their expressional patterns were also systematically analyzed. Additionally, subcellular localization analysis showed that they are mainly localized in the nucleus, of which a SEPALLATA3 (SEP3) homolog NnMADS14 was proven to be involved in the floral organogenesis. CONCLUSION: These results provide some fundamental information about the MADS-box gene family and their functions, which might be helpful in not only understanding the mechanisms of floral organogenesis but also breeding of high ornamental value cultivars in lotus.

Proteomic analysis showing the signaling pathways involved in the rhizome enlargement process in Nelumbo nucifera.

BACKGROUND: Rhizome is the storage underground stem of lotus (Nelumbo nucifera), which is enlarged before winter season and could be used for asexual propagation. In addition, the enlarged rhizome is a nutritional vegetable with abundant starch, proteins, and vitamins. Enlargement of lotus rhizome is not only significance for itself to survive from the cold winter, but also important for its economic value. RESULTS: To explore the mechanism underlying its enlargement, integrative analyses of morphology, physiology and proteomics were conducted on the rhizome at stolon, middle, and enlarged stages. Morphological observation and physiological analyses showed that rhizomes were gradually enlarged during this process, in which the starch accumulation was also initiated. Quantitative proteomic analysis on the rhizomes at these three stages identified 302 stage-specific proteins (SSPs) and 172 differently expressed proteins (DEPs), based on which GO and KEGG enrichment analyses were conducted. The results indicated that light and auxin signal might be transduced through secondary messenger Ca2+, and play important roles in lotus rhizome enlargement. CONCLUSION: These results will provide new insights into understanding the mechanism of lotus rhizome enlargement. Meanwhile, some candidate genes might be useful for further studies on this process, as well as breeding of rhizome lotus.

Nitrogen Fertilizer Induced Alterations in The Root Proteome of Two Rice Cultivars.

Nitrogen (N) is an essential nutrient for plants and a key limiting factor of crop production. However, excessive application of N fertilizers and the low nitrogen use efficiency (NUE) have brought in severe damage to the environment. Therefore, improving NUE is urgent and critical for the reductions of N fertilizer pollution and production cost. In the present study, we investigated the effects of N nutrition on the growth and yield of the two rice (Oryza sativa L.) cultivars, conventional rice Huanghuazhan and indica hybrid rice Quanliangyou 681, which were grown at three levels of N fertilizer (including 135, 180 and 225 kg/hm2, labeled as N9, N12, N15, respectively). Then, a proteomic approach was employed in the roots of the two rice cultivars treated with N fertilizer at the level of N15. A total of 6728 proteins were identified, among which 6093 proteins were quantified, and 511 differentially expressed proteins were found in the two rice cultivars after N fertilizer treatment. These differentially expressed proteins were mainly involved in ammonium assimilation, amino acid metabolism, carbohydrate metabolism, lipid metabolism, signal transduction, energy production/regulation, material transport, and stress/defense response. Together, this study provides new insights into the regulatory mechanism of nitrogen fertilization in cereal crops.

The Latest Studies on Lotus (Nelumbo nucifera)-an Emerging Horticultural Model Plant.

Lotus (Nelumbo nucifera) is a perennial aquatic basal eudicot belonging to a small family Nelumbonaceace, which contains only one genus with two species. It is an important horticultural plant, with its uses ranging from ornamental, nutritional to medicinal values, and has been widely used, especially in Southeast Asia. Recently, the lotus obtained a lot of attention from the scientific community. An increasing number of research papers focusing on it have been published, which have shed light on the mysteries of this species. Here, we comprehensively reviewed the latest advancement of studies on the lotus, including phylogeny, genomics and the molecular mechanisms underlying its unique properties, its economic important traits, and so on. Meanwhile, current limitations in the research of the lotus were addressed, and the potential prospective were proposed as well. We believe that the lotus will be an important model plant in horticulture with the generation of germplasm suitable for laboratory operation and the establishment of a regeneration and transformation system.

Genome-Wide DNA Methylation Profiling in the Lotus (Nelumbo nucifera) Flower Showing its Contribution to the Stamen Petaloid.

DNA methylation is a vital epigenetic modification. Methylation has a significant effect on the gene expression influencing the regulation of different physiological processes. Current studies on DNA methylation have been conducted on model plants. Lotus (Nelumbo nucifera) is a basic eudicot exhibiting variations during development, especially in flower formation. DNA methylation profiling was conducted on different flower tissues of lotuses through whole genome bisulfite sequencing (WGBS) to investigate the effects of DNA methylation on its stamen petaloid. A map of methylated cytosines at the single base pair resolution for the lotus was constructed. When the stamen was compared with the stamen petaloid, the DNA methylation exhibited a global decrease. Genome-wide relationship analysis between DNA methylation and gene expression identified 31 different methylation region (DMR)-associated genes, which might play crucial roles in floral organ formation, especially in the stamen petaloid. One out of 31 DMR-associated genes, NNU_05638 was homolog with Plant U-box 33 (PUB33). The DNA methylation status of NNU_05638 promoter was distinct in three floral organs, which was confirmed by traditional bisulfite sequencing. These results provide further insights about the regulation of stamen petaloids at the epigenetic level in lotus.

The Rice Alpha-Amylase, Conserved Regulator of Seed Maturation and Germination.

Alpha-amylase, the major form of amylase with secondary carbohydrate binding sites, is a crucial enzyme throughout the growth period and life cycle of angiosperm. In rice, alpha-amylase isozymes are critical for the formation of the storage starch granule during seed maturation and motivate the stored starch to nourish the developing seedling during seed germination which will directly affect the plant growth and field yield. Alpha-amylase has not yet been studied intensely to understand its classification, structure, expression trait, and expression regulation in rice and other crops. Among the 10-rice alpha-amylases, most were exclusively expressed in the developing seed embryo and induced in the seed germination process. During rice seed germination, the expression of alpha-amylase genes is known to be regulated negatively by sugar in embryos, however positively by gibberellin (GA) in endosperm through competitively binding to the specific promoter domain; besides, it is also controlled by a series of other abiotic or biotic factors, such as salinity. In this review, we overviewed the research progress of alpha-amylase with focus on seed germination and reflected on how in-depth work might elucidate its regulation and facilitate crop breeding as an efficient biomarker.

Transcriptomic analysis identifies the key genes involved in stamen petaloid in lotus (Nelumbo nucifera).

BACKGROUND: Flower morphology, a phenomenon regulated by a complex network, is one of the vital ornamental features in Nelumbo nucifera. Stamen petaloid is very prevalent in lotus flowers. However, the mechanism underlying this phenomenon is still obscure. RESULTS: Here, the comparative transcriptomic analysis was performed among petal, stamen petaloid and stamen through RNA-seq. Using pairwise comparison analysis, a large number of genes involved in hormonal signal transduction pathways and transcription factors, especially the MADS-box genes, were identified as candidate genes for stamen petaloid in lotus. CONCLUSIONS: Taken together, these results provide an insight into the molecular networks underlying lotus floral organ development and stamen petaloid.

Proteomics analysis identified a DRT protein involved in arsenic resistance in Populus.

KEY MESSAGE: A DRT protein was identified and proved to be involved in the poplar arsenic resistance through comparative proteomics analysis between arsenic sensitive and resistant cultivars. Arsenic pollution in soil has been a serious problem all over the world. It is very important to dissect plants arsenic stress-response mechanisms in phytoremediation. In this study, arsenate-tolerant Populus deltoides cv. 'zhonglin 2025' and arsenate-sensitive Populus × euramericana cv. 'I-45/51' were screened from 10 poplar varieties. Systematic comparisons between these two cultivars demonstrated that 'zhonglin 2025' exhibited slighter morphological and structural injury, lower ROS and MDA accumulation, and higher photosynthesis and ROS scavenging ability under arsenate stress, compared with 'I-45/51'. Through comparative proteomics analysis, we detected that most of the identified arsenate-responsive proteins were stress and defense related. Among these proteins, PdDRT102 was found to be only highly induced in 'zhonglin 2025' under arsenate stress. Heterologous over-expression of PdDRT102 in Arabidopsis conferred to enhanced tolerance to arsenate and sodium chloride. PdDRT102 localizes to the plasma membrane and the nucleus in Arabidopsis. Interestingly, the remarkably increased fluorescence protein signals in the nucleus were found during arsenate stress. Together, these results not only provided an overall understanding on poplar response to arsenate stress, but also revealed that DRT102 protein might involve in protecting poplar against this stress.

A proteomic analysis of salt stress response in seedlings of two African rice cultivars.

Salt stress is one of the key abiotic stresses threatening future agricultural production and natural ecosystems. This study investigates the salt stress response of two rice seedlings, which were screened from 28 Kenya rice cultivars. A proteomic analysis was carried out and Mapman bin codes employed in protein function categorization. Proteins in the redox, stress, and signaling categories were identified, and whose expression differed between the salt tolerant and the salt sensitive samples employed in the present study. 104 and 102 root proteins were observed as significantly altered during salt stress in the tolerant and sensitive samples, respectively and 13 proteins were commonly expressed. Among the 13 proteins, ketol-acid reductoisomerase protein was upregulated in both 1 and 3days of salt treatment in the tolerant sample, while it was down-regulated in both 1 and 3days of salt treatment in the sensitive sample. Actin-7, tubulin alpha, V-type proton ATPase, SOD (Cu-Zn), SOD (Mn), and pyruvate decarboxylase were among the observed salt-induced proteins. In general, this study improves our understanding about salt stress response mechanisms in rice.