DIA-Based Quantitative Proteomics Reveals the Protein Regulatory Networks of Floral Thermogenesis in Nelumbo nucifera.

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.

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.

Integrated Omics Analyses Identify Key Pathways Involved in Petiole Rigidity Formation in Sacred Lotus.

Sacred lotus (Nelumbo nucifera Gaertn.) is a relic aquatic plant with two types of leaves, which have distinct rigidity of petioles. Here we assess the difference from anatomic structure to the expression of genes and proteins in two petioles types, and identify key pathways involved in petiole rigidity formation in sacred lotus. Anatomically, great variation between the petioles of floating and vertical leaves were observed. The number of collenchyma cells and thickness of xylem vessel cell wall was higher in the initial vertical leaves' petiole (IVP) compared to the initial floating leaves' petiole (IFP). Among quantified transcripts and proteins, 1021 and 401 transcripts presented 2-fold expression increment (named DEGs, genes differentially expressed between IFP and IVP) in IFP and IVP, 421 and 483 proteins exhibited 1.5-fold expression increment (named DEPs, proteins differentially expressed between IFP and IVP) in IFP and IVP, respectively. Gene function and pathway enrichment analysis displayed that DEGs and DEPs were significantly enriched in cell wall biosynthesis and lignin biosynthesis. In consistent with genes and proteins expressions in lignin biosynthesis, the contents of lignin monomers precursors were significantly different in IFP and IVP. These results enable us to understand lotus petioles rigidity formation better and provide valuable candidate genes information on further investigation.

Small RNA and Transcriptome Sequencing Reveals miRNA Regulation of Floral Thermogenesis in Nelumbo nucifera.

The sacred lotus (Nelumbo nucifera Gaertn.) can produce heat autonomously and maintain a relatively stable floral chamber temperature for several days when blooming. Floral thermogenesis is critical for flower organ development and reproductive success. However, the regulatory role of microRNA (miRNA) underlying floral thermogenesis in N. nucifera remains unclear. To comprehensively understand the miRNA regulatory mechanism of thermogenesis, we performed small RNA sequencing and transcriptome sequencing on receptacles from five different developmental stages. In the present study, a total of 172 known miRNAs belonging to 39 miRNA families and 126 novel miRNAs were identified. Twenty-nine thermogenesis-related miRNAs and 3024 thermogenesis-related mRNAs were screened based on their expression patterns. Of those, seventeen differentially expressed miRNAs (DEMs) and 1765 differentially expressed genes (DEGs) had higher expression during thermogenic stages. The upregulated genes in the thermogenic stages were mainly associated with mitochondrial function, oxidoreductase activity, and the energy metabolism process. Further analysis showed that miR156_2, miR395a_5, miR481d, and miR319p may play an important role in heat-producing activity by regulating cellular respiration-related genes. This study provides comprehensive miRNA and mRNA expression profile of receptacle during thermogenesis in N. nucifera, which advances our understanding on the regulation of floral thermogenesis mediated by miRNA.

Transcriptome analysis of miRNAs expression reveals novel insights into adventitious root formation in lotus (Nelumbo nucifera Gaertn.).

MicroRNA (miRNA)-regulated gene expression plays an important role in various plant metabolic processes. Although adventitious roots are critical to plant growth in lotus, the role of miRNA in AR formation remains unclear. Expression profiling of miRNAs was carried out during three different developmental stages of ARs in lotus: no induction of AR stage, initial stage of ARs, and maximum number of ARs. These data are referenced with the whole lotus genome as already identified through high-throughput tag-sequencing. 1.3 × 107 tags were achieved, of which 11,035,798, 11,436,062, and 12,542,392 clean tags were obtained from each stage, respectively. miRNA analysis revealed that miRNAs were less than 10% among all small RNAs. In total, 310 miRNAs (240 up-regulated and 70 down-regulated miRNAs) exhibited expression changes from the no induction stage to the initial stage. Moreover, expression of 140 miRNAs was increased and that of 123 miRNAs was decreased between the initial and maximum AR stages, mostly by ~ - 4-4-fold. miRNAs involved in metabolic pathways differed between the initial stage/no induction stage and the maximum number stage/initial stage. Several miRNAs in the initial stage/no induction stage were related to plant hormone metabolism and pyruvate and MAPK pathways, while major miRNAs in the maximum number stage/initial stage were involved in carbohydrate metabolism. All differentially expressed miRNAs associated with AR formation from the initial stage to maximum stage were also analyzed. The expression of 16 miRNAs was determined using qRT-PCR. This work provides a general insight into miRNA regulation during AR formation in lotus.

A comparative proteomic analysis for adventitious root formation in lotus root (Nelumbo nucifera Gaertn).

Adventitious roots (ARs) directly affect lotus seedling growth and product quality because principal root is not well developed. However, the details of AR formation at the molecular level have not been determined in lotus. Therefore, three stages were chosen to identify the change of proteins abundant during rhizome formation, using isobaric tags for relative and absolute quantization coupled with liquid chromatography-tandem mass spectrometry to gain insight into the molecular mechanisms involved in AR formation. We totally obtained 323,375 spectra during AR formation. After filtering to eliminate low-scoring spectra, 66,943 spectra, including 53,106 unique spectra, were identified. These unique spectra matched 28,905 peptides, including 24,992 unique peptides, which were assembled into 6686 proteins. In the C0/C1 and C1/C2 stages, 66 and 32 proteins showed enhanced abundance, and 173 and 73 proteins showed decreased abundance, respectively. Seventeen important AR formation-related proteins from the three stages were identified, and the expressions of nine genes from the above-identified proteins were assessed by qRT-PCR. This article provides a comprehensive understanding of the changes in metabolism during AR formation, and is helpful to accelerate the progress of breeding in fulture in lotus root.

Analysis of Isoquinoline Alkaloid Composition and Wound-Induced Variation in Nelumbo Using HPLC-MS/MS.

Alkaloids are the most relevant bioactive components in lotus, a traditional herb in Asia, but little is known about their qualitative and quantitative distributions. Here, we report on the alkaloid composition in various lotus organs. Lotus laminae and embryos are rich in isoquinoline alkaloids, whereas petioles and rhizomes contain trace amounts of alkaloids. Wide variation of alkaloid accumulation in lamina and embryo was observed among screened genotypes. In laminae, alkaloid accumulation increases during early developmental stages, reaches the highest level at full size stage, and then decreases slightly during senescence. Vegetative and embryogenic tissues accumulate mainly aporphine-type and bisbenzylisoquinoline-type alkaloids, respectively. Bisbenzylisoquinoline-type alkaloids may be synthesized mainly in lamina and then transported into embryo via latex through phloem translocation. In addition, mechanical wounding was shown to induce significant accumulation of specific alkaloids in lotus leaves.

Transcriptomic Analysis of the Regulation of Rhizome Formation in Temperate and Tropical Lotus (Nelumbo nucifera).

Rhizome is the storage organ of lotus derived from modified stems. The development of rhizome is a complex process and depends on the balanced expression of the genes that is controlled by environmental and endogenous factors. However, little is known about the mechanism that regulates rhizome girth enlargement. In this study, using RNA-seq, transcriptomic analyses were performed at three rhizome developmental stages-the stolon, middle swelling and later swelling stage -in the cultivars 'ZO' (temperate lotus with enlarged rhizome) and 'RL' (tropical lotus with stolon). About 348 million high-quality reads were generated, and 88.5% of the data were mapped to the reference genome. Of 26783 genes identified, 24069 genes were previously predicted in the reference, and 2714 genes were novel transcripts. Moreover, 8821 genes were differentially expressed between the cultivars at the three stages. Functional analysis identified that these genes were significantly enriched in pathways carbohydrate metabolism and plant hormone signal transduction. Twenty-two genes involved in photoperiod pathway, starch metabolism and hormone signal transduction were candidate genes inducing rhizome girth enlargement. Comparative transcriptomic analysis detected several differentially expressed genes and potential candidate genes required for rhizome girth enlargement, which lay a foundation for future studies on molecular mechanisms underlying rhizome formation.

Cloning and expression of an APETALA1-like gene from Nelumbo nucifera.

The objective of this study was to clone the full-length cDNA of the APETALA1 (AP1) gene from lotus and analyze its sequence and expression pattern. The full-length cDNA sequence of the NnAP1 gene was amplified from the petals of Nelumbo nucifera 'Hongxia' using RT-PCR and rapid amplification of cDNA ends. Bioinformatic methods were used to analyze the sequence characteristics of the gene. Quantitative real-time PCR methods were used to investigate the expression pattern of NnAP1 in various organs and during different developmental stages. The cloned full-length NnAP1 cDNA (GenBank accession No. KF361315) was 902 bp, containing a 795-bp open reading frame encoding 264 amino acids with a relative molecular mass of 30,288.4 and an isoelectric point of 9.13. NnAP1 had a MADS-box domain and a K-box domain, which is typical of the SQUA/AP1 gene family. A protein sequence identity search showed that NnAP1 was 75-96% similar to other plant AP1s. Phylogenetic tree analysis indicated that NnAP1 was very closely related to AP1 of Glycine max, suggesting that they shared the same protein ancestor. Quantitative real-time PCR analysis showed that NnAP1 was expressed in various organs during different developmental stages; it had the highest expression in blooming flowers and had trace expression in the young vegetative and flower senescence stages. Our analysis suggests that NnAP1 plays an important role in controlling floral meristem identity and floral organ formation.

Characterization of flower-bud transcriptome and development of genic SSR markers in Asian lotus (Nelumbo nucifera Gaertn.).

BACKGROUND: Asian lotus (Nelumbo nucifera Gaertn.) is the national flower of India, Vietnam, and one of the top ten traditional Chinese flowers. Although lotus is highly valued for its ornamental, economic and cultural uses, genomic information, particularly the expressed sequence based (genic) markers is limited. High-throughput transcriptome sequencing provides large amounts of transcriptome data for promoting gene discovery and development of molecular markers. RESULTS: In this study, 68,593 unigenes were assembled from 1.34 million 454 GS-FLX sequence reads of a mixed flower-bud cDNA pool derived from three accessions of N. nucifera. A total of 5,226 SSR loci were identified, and 3,059 primer pairs were designed for marker development. Di-nucleotide repeat motifs were the most abundant type identified with a frequency of 65.2%, followed by tri- (31.7%), tetra- (2.1%), penta- (0.5%) and hexa-nucleotide repeats (0.5%). A total of 575 primer pairs were synthesized, of which 514 (89.4%) yielded PCR amplification products. In eight Nelumbo accessions, 109 markers were polymorphic. They were used to genotype a sample of 44 accessions representing diverse wild and cultivated genotypes of Nelumbo. The number of alleles per locus varied from 2 to 9 alleles and the polymorphism information content values ranged from 0.6 to 0.9. We performed genetic diversity analysis using 109 polymorphic markers. A UPGMA dendrogram was constructed based on Jaccard's similarity coefficients revealing distinct clusters among the 44 accessions. CONCLUSIONS: Deep transcriptome sequencing of lotus flower buds developed 3,059 genic SSRs, making a significant addition to the existing SSR markers in lotus. Among them, 109 polymorphic markers were successfully validated in 44 accessions of Nelumbo. This comprehensive set of genic SSR markers developed in our study will facilitate analyses of genetic diversity, construction of linkage maps, gene mapping, and marker-assisted selection breeding for lotus.

Comparative analysis of antioxidant activity and functional components of the ethanol extract of lotus (Nelumbo nucifera) from various growing regions.

The variations in antioxidant activity and concentration of functional components in the ethanol extracts of lotus seeds and rhizomes based on the growing region and dryness were investigated. Free radical scavenging activity, total phenolic and flavonoid content, and concentration of several specific flavonoids and alkaloids in the ethanol extracts of lotus were measured. Antioxidant activity and its correlative total phenolic content varied characteristically depending on the growing region and dryness. High-perfomance liquid chromatography analysis showed that the ethanol extracts of lotus seeds from Vietnam (Ho Chi Minh City), raw rhizomes from Korea (Siheung), and dried rhizomes from Japan (Nigata) had the greatest specific flavonoid content. The ethanol extracts of seeds from China (Hubei), raw rhizomes from Japan (Nigata), and dried rhizomes from Korea (Siheung) had the greatest specific alkaloid content. Astragaline, rutin, isoquercetin, nuciferine, dauricine, isoliensinine, and neferine were identified in lotus rhizomes for the first time in this study.

The sacred lotus genome provides insights into the evolution of flowering plants.

Sacred lotus (Nelumbo nucifera) is an ornamental plant that is also used for food and medicine. This basal eudicot species is especially important from an evolutionary perspective, as it occupies a critical phylogenetic position in flowering plants. Here we report the draft genome of a wild strain of sacred lotus. The assembled genome is 792 Mb, which is approximately 85-90% of genome size estimates. We annotated 392 Mb of repeat sequences and 36,385 protein-coding genes within the genome. Using these sequence data, we constructed a phylogenetic tree and confirmed the basal location of sacred lotus within eudicots. Importantly, we found evidence for a relatively recent whole-genome duplication event; any indication of the ancient paleo-hexaploid event was, however, absent. Genomic analysis revealed evidence of positive selection within 28 embryo-defective genes and one annexin gene that may be related to the long-term viability of sacred lotus seed. We also identified a significant expansion of starch synthase genes, which probably elevated starch levels within the rhizome of sacred lotus. Sequencing this strain of sacred lotus thus provided important insights into the evolution of flowering plant and revealed genetic mechanisms that influence seed dormancy and starch synthesis.

Genome-Wide Analysis of Differentially Expressed Genes Relevant to Rhizome Formation in Lotus Root (Nelumbo nucifera Gaertn).

Lotus root is a popular wetland vegetable which produces edible rhizome. At the molecular level, the regulation of rhizome formation is very complex, which has not been sufficiently addressed in research. In this study, to identify differentially expressed genes (DEGs) in lotus root, four libraries (L1 library: stolon stage, L2 library: initial swelling stage, L3 library: middle swelling stage, L4: later swelling stage) were constructed from the rhizome development stages. High-throughput tag-sequencing technique was used which is based on Solexa Genome Analyzer Platform. Approximately 5.0 million tags were sequenced, and 4542104, 4474755, 4777919, and 4750348 clean tags including 151282, 137476, 215872, and 166005 distinct tags were obtained after removal of low quality tags from each library respectively. More than 43% distinct tags were unambiguous tags mapping to the reference genes, and 40% were unambiguous tag-mapped genes. From L1, L2, L3, and L4, total 20471, 18785, 23448, and 21778 genes were annotated, after mapping their functions in existing databases. Profiling of gene expression in L1/L2, L2/L3, and L3/L4 libraries were different among most of the selected 20 DEGs. Most of the DEGs in L1/L2 libraries were relevant to fiber development and stress response, while in L2/L3 and L3/L4 libraries, major of the DEGs were involved in metabolism of energy and storage. All up-regulated transcriptional factors in four libraries and 14 important rhizome formation-related genes in four libraries were also identified. In addition, the expression of 9 genes from identified DEGs was performed by qRT-PCR method. In a summary, this study provides a comprehensive understanding of gene expression during the rhizome formation in lotus root.

Isolation and functional characterization of a salt responsive transcriptional factor, LrbZIP from lotus root (Nelumbo nucifera Gaertn).

Basic leucine zipper transcription factor (bZIP) is involved in signaling transduction for various stress responses. Here we reported a bZIP transcription factor (accession: JX887153) isolated from a salt-resistant lotus root using cDNA-AFLP approach with RT-PCR and RACE-PCR method. Full-length cDNA which consisted of a single open reading frame encoded a putative polypeptide of 488 amino acids. On the basis of 78, 76, and 75 % sequence similarity with the bZIPs from Medicago truncatula (XP_003596814.1), Carica papaya (ABS01351.1) and Arabidopsis thaliana (NP_563810.2), we designed it as LrbZIP. Semi quantitative RT-PCR results, performed on the total RNA extracted from tips of lotus root, showed that LrbZIP expression was increased with 250 mM NaCl treatment for 18 h. Effects of low temperature on the expression of LrbZIP was also studied, and its expression was significantly enhanced with a 4 °C treatment for 12 h. In addition, LrbZIP expression was strongly induced by treatment with exogenous 100 µM ABA. To evaluate its function across the species, tobacco (Nicotiana tabacum L.) was transformed with LrbZIP in a binary vector construct. Transgenic plants exhibited higher resistance as compared with the control according to the results of the root growth, chlorophyll content and electrolyte leakage when exposed to NaCl treatment. In addition, LrCDPK2, LrLEA, and TPP also showed enhanced expression in the transgenic plants. Overall, expression of LrbZIP was probably very important for salt-resistant lotus root to survive through salt stress.

Genetic linkage maps for Asian and American lotus constructed using novel SSR markers derived from the genome of sequenced cultivar.

BACKGROUND: The genus Nelumbo Adans. comprises two living species, N. nucifera Gaertan. (Asian lotus) and N. lutea Pers. (American lotus). A genetic linkage map is an essential resource for plant genetic studies and crop improvement but has not been generated for Nelumbo. We aimed to develop genomic simple sequence repeat (SSR) markers from the genome sequence and construct two genetic maps for Nelumbo to assist genome assembly and integration of a genetic map with the genome sequence. RESULTS: A total of 86,089 SSR motifs were identified from the genome sequences. Di- and tri-nucleotide repeat motifs were the most abundant, and accounted for 60.73% and 31.66% of all SSRs, respectively. AG/GA repeats constituted 51.17% of dinucleotide repeat motifs, followed by AT/TA (44.29%). Of 500 SSR primers tested, 386 (77.20%) produced scorable alleles with an average of 2.59 per primer, and 185 (37.00%) showed polymorphism among two parental genotypes, N. nucifera 'Chinese Antique' and N. lutea 'AL1', and six progenies of their F1 population. The normally segregating markers, which comprised 268 newly developed SSRs, 37 previously published SSRs and 53 sequence-related amplified polymorphism markers, were used for genetic map construction. The map for Asian lotus was 365.67 cM with 47 markers distributed in seven linkage groups. The map for American lotus was 524.51 cM, and contained 177 markers distributed in 11 genetic linkage groups. The number of markers per linkage group ranged from three to 34 with an average genetic distance of 3.97 cM between adjacent markers. Moreover, 171 SSR markers contained in linkage groups were anchored to 97 genomic DNA sequence contigs of 'Chinese Antique'. The 97 contigs were merged into 60 scaffolds. CONCLUSION: Genetic mapping of SSR markers derived from sequenced contigs in Nelumbo enabled the associated contigs to be anchored in the linkage map and facilitated assembly of the genome sequences of 'Chinese Antique'. The present study reports the first construction of genetic linkage maps for Nelumbo, which can serve as reference linkage maps to accelerate characterization germplasm, genetic mapping for traits of economic interest, and molecular breeding with marker-assisted selection.

Identification and expression of an APETALA2-like gene from Nelumbo nucifera.

Arabidopsis transcription factor APETALA2 (AP2) controls multiple aspects of plant growth and development, including seed development, stem cell maintenance, and specification of floral organ identity. Based on sequence similar of Arabidopsis AP2 and its homologues genes from other plant species, degenerate RT-PCR and rapid amplification of cDNA ends assay were used to clone AP2 genes from lotus (Nelumbo nucifera). A 2,048-bp cDNA fragment was obtained, which contains a 1,536-bp open reading frame encoding a protein of 511 amino acids. The protein contains two AP2 domains that are conserved in AP2 proteins from other plant species, thus was named as N. nucifera APETALA2 (NnAP2). Quantitative RT-PCR revealed that NnAP2 gene was expressed in flowers, roots, leaves, and stems of N. nucifera, with flowers which have the highest transcript levels. Further analysis showed that in all five lotus cultivars examined, including "Zhongguogudailian," "Yaoniangyujiao," "Jinxia," "Hongtailian," and "Yiliangqianban," petals always have the highest expression levels when compared with the other four flower organs, though the number of petals in these cultivars ranged from simple to thousands. However, NnAP2 expression level in four nonsimple petal flower cultivars was higher than that in the simple petal flower cultivar Zhongguogudailian, indicating that NnAP2 may play a role in specification of petal identity during the evolutionary process of the ancient species N. nucifera.

Proteomic and functional analyses of Nelumbo nucifera annexins involved in seed thermotolerance and germination vigor.

Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. Although there is increasing evidence implicating their importance in plant stress responses, their functions in seeds remain to be further studied. In this study, we identified a heat-induced annexin, NnANN1, from the embryonic axes of sacred lotus (Nelumbo nucifera Gaertn.) using comparative proteomics approach. Moreover, the expression of NnANN1 increased considerably in response to high-temperature treatment. Quantitative real-time PCR (qRT-PCR) revealed that the transcripts of NnANN1 were detected predominantly during seed development and germination in sacred lotus, implicating a role for NnANN1 in plant seeds. Ectopic expression of NnANN1 in Arabidopsis resulted in enhanced tolerance to heat stress in transgenic seeds. In addition, compared to the wild-type seeds, transgenic seeds ectopically expressing NnANN1 exhibited improved resistance to accelerated aging treatment used for assessing seed vigor. Furthermore, transgenic seeds showed enhanced peroxidase activities, accompanied with reduced lipid peroxidation and reduced ROS release levels compared to the wild-type seeds. Taken together, these results indicate that NnANN1 plays an important role in seed thermotolerance and germination vigor.

Remediation of cadmium contaminated irrigation and drinking water: a large scale approach.

Cadmium is one of the most troublesome toxic heavy metals. It accumulates in the water reservoirs and agricultural soil as a result of intensive use of Cd contaminated phosphate fertilizers, e.g. in agriculture in the North Central Province (NCP) of Sri Lanka. The hyper-accumulator Thlaspi caerulescens, accumulates up to 1000 ppm Cd in shoots without exhibiting toxicity symptoms. The storage rhizomes of year old Nelumbo nucifera (lotus) natural vegetation in water reservoirs in NCP accumulated 253+/-12 mg Cd/kg. Seedlings of lotus grown in 5% Hoagland's solution at 0.75, 1.0 and 1.25 ppm cadmium sulphate showed a significant increase in Cd removal of 0.0334-0.121 ppm/week. However the removal rate of Cd from water failed to increase any further at higher concentrations of Cd in water. The slow growth rate and low rate of phytoextraction demands a more effective but an affordable method of remediation in order to combat the prevailing elevated cadmium levels in NCP that causes chronic renal failure (CRF). We have developed a large scale filtering device using rice husk. We have achieved successful results in sequestering Cd using raw rice husk as well as amorphous silica derived from rice husk.

Synchronicity of thermogenic activity, alternative pathway respiratory flux, AOX protein content, and carbohydrates in receptacle tissues of sacred lotus during floral development.

The relationships between heat production, alternative oxidase (AOX) pathway flux, AOX protein, and carbohydrates during floral development in Nelumbo nucifera (Gaertn.) were investigated. Three distinct physiological phases were identified: pre-thermogenic, thermogenic, and post-thermogenic. The shift to thermogenic activity was associated with a rapid, 10-fold increase in AOX protein. Similarly, a rapid decrease in AOX protein occurred post-thermogenesis. This synchronicity between AOX protein and thermogenic activity contrasts with other thermogenic plants where AOX protein increases some days prior to heating. AOX protein in thermogenic receptacles was significantly higher than in post-thermogenic and leaf tissues. Stable oxygen isotope measurements confirmed that the increased respiratory flux supporting thermogenesis was largely via the AOX, with little or no contribution from the cytochrome oxidase pathway. During the thermogenic phase, no significant relationship was found between AOX protein content and either heating or AOX flux, suggesting that regulation is likely to be post-translational. Further, no evidence of substrate limitation was found; starch accumulated during the early stages of floral development, peaking in thermogenic receptacles, before declining by 89% in post-thermogenic receptacles. Whilst coarse regulation of AOX flux occurs via protein synthesis, the ability to thermoregulate probably involves precise regulation of AOX protein, most probably by effectors such as alpha-keto acids.