Ethnobotanical History: Duckweeds in Different Civilizations.

This presentation examines the history of duckweeds in Chinese, Christian, Greek, Hebrew, Hindu, Japanese, Maya, Muslim, and Roman cultures and details the usage of these diminutive freshwater plants from ancient times through the Middle Ages. We find that duckweeds were widely distributed geographically already in antiquity and were integrated in classical cultures in the Americas, Europe, the Near East, and the Far East 2000 years ago. In ancient medicinal sources, duckweeds are encountered in procedures, concoctions, and incantations involving the reduction of high fever. In this regard, we discuss a potential case of ethnobotanical convergence between the Chinese Han and Classical Maya cultures. Duckweeds played a part in several ancient rituals. In one, the unsuitability of its roots to serve as a wick for Sabbath oil lamps. In another reference to its early use as human food during penitence. In a third, a prominent ingredient in a medicinal incantation, and in a fourth, as a crucial element in ritual body purifications. Unexpectedly, it emerged that in several ancient cultures, the floating duckweed plant featured prominently in the vernacular and religious poetry of the day.

Novel Mutation in the Acetohydroxyacid Synthase (AHAS), Gene Confers Imidazolinone Resistance in Chickpea Cicer arietinum L. Plants.

Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicides but not to other groups of AHAS inhibitors, is described in the AHAS1 protein of EMS-mutagenized chickpea line M2033.

Return of the Lemnaceae: duckweed as a model plant system in the genomics and postgenomics era.

The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society's pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.

Identification, Phylogeny, and Comparative Expression of the Lipoxygenase Gene Family of the Aquatic Duckweed, Spirodela polyrhiza, during Growth and in Response to Methyl Jasmonate and Salt.

Lipoxygenases (LOXs) (EC 1.13.11.12) catalyze the oxygenation of fatty acids and produce oxylipins, including the plant hormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA). Little information is available about the LOX gene family in aquatic plants. We identified a novel LOX gene family comprising nine LOX genes in the aquatic plant Spirodela polyrhiza (greater duckweed). The reduced anatomy of S. polyrhiza did not lead to a reduction in LOX family genes. The 13-LOX subfamily, with seven genes, predominates, while the 9-LOX subfamily is reduced to two genes, an opposite trend from known LOX families of other plant species. As the 13-LOX subfamily is associated with the synthesis of JA/MeJA, its predominance in the Spirodela genome raises the possibility of a higher requirement for the hormone in the aquatic plant. JA-/MeJA-based feedback regulation during culture aging as well as the induction of LOX gene family members within 6 h of salt exposure are demonstrated.

Photosystem-II D1 protein mutants of Chlamydomonas reinhardtii in relation to metabolic rewiring and remodelling of H-bond network at QB site.

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency - HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, ß-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the QB site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.

Nutrient Value of Leaf vs. Seed.

Major differences stand out between edible leaves and seeds in protein quality, vitamin, and mineral concentrations and omega 6/omega 3 fatty acid ratios. Data for seeds (wheat, rice, corn, soy, lentil, chick pea) are compared with corresponding data for edible green leaves (kale, spinach, broccoli, duckweed). An x/y representation of data for lysine and methionine content highlights the group differences between grains, pulses, leafy vegetables, and animal foods. Leaves come out with flying colors in all these comparisons. The perspective ends with a discussion on "So why do we eat mainly seeds?"

Expression of flowering locus T2 transgene from Pyrus communis L. delays dormancy and leaf senescence in Malus×domestica Borkh, and causes early flowering in tobacco.

Annual and perennial plants represent two different evolutionary strategies based on differential synchronization of their reproductive development. The mobile signal protein FLOWERING LOCUS T (FT) plays a central role in mediating the onset of reproduction in both plant types. Two novel FT-like genes from pear (Pyrus communis)-PcFT1 and PcFT2-were isolated, and their expression profiles were determined for one annual cycle. The effects of PcFT2 on flowering were investigated in annual (tobacco) and perennial (apple) plants by means of grafting and generating transgenic plants. Long-distance graft transmission of PcFT2 in both annual and perennial plants was confirmed using a 35S::PcFT2-YFP construct. Ectopic overexpression of PcFT2 caused early flowering in tobacco but not in apple. Transgenic apples were less sensitive to short-day-induced dormancy, and this phenotype was also observed in wild-type apples grafted onto the transgenic plants. Comparison of PcFT2 protein structure to the paralogous FT proteins from apple and pear showed alterations that could influence protein structure and thus the florigen-activation complex. PcFT2 protein seems to function by promoting flowering as all other FT proteins in the annual plant tobacco while in the perennial plant apple PcFT2 does not promote flowering but delays senescence. This observation may hint to a modified function of FT2 in perennial plants.

Genome-wide computational determination of the human metalloproteome.

Accurate prediction of protein function in humans is important for understanding biological processes at the molecular level in biomedicine and drug design. Over a third of proteins are commonly held to bind metal, and ∼10% of human proteins, to bind zinc. Therefore, an initial step in protein function prediction frequently involves predicting metal ion binding. In recent years, methods have been developed to predict a set of residues in 3D space forming the metal-ion binding site, often with a high degree of accuracy. Here, using extensions of these methods, we provide an extensive list of human proteins and their putative metal ion binding site residues, using translated gene sequences derived from the complete, resolved human genome. Under conditions of ∼90% selectivity, over 900 new human putative metal ion binding proteins are identified. A statistical analysis of resolved metal ion binding sites in the human metalloproteome is furnished and the importance of remote homology analysis is demonstrated. As an example, a novel metal-ion binding site involving a complex of a botulinum substrate with its inhibitor is presented. On the basis of the location of the predicted site and the interactions of the contacting residues at the complex interface, we postulate that metal ion binding in this region could influence complex formation and, consequently, the functioning of the protein. Thus, this work provides testable hypotheses about novel functions of known proteins.

Structure of ALD1, a plant-specific homologue of the universal diaminopimelate aminotransferase enzyme of lysine biosynthesis.

Diaminopimelate aminotransferase (DAP-AT) is an enzyme in the lysine-biosynthesis pathway. Conversely, ALD1, a close homologue of DAP-AT in plants, uses lysine as a substrate in vitro. Both proteins require pyridoxal-5'-phosphate (PLP) for their activity. The structure of ALD1 from the flowering plant Arabidopsis thaliana (AtALD1) was solved at a resolution of 2.3 Å. Comparison of AtALD1 with the previously solved structure of A. thaliana DAP-AT (AtDAP-AT) revealed similar interactions with PLP despite sequence differences within the PLP-binding site. However, sequence differences between the binding site of AtDAP-AT for malate, a purported mimic of substrate binding, and the corresponding site in AtALD1 led to different interactions. This suggests that either the substrate itself, or the substrate-binding mode, differs in the two proteins, supporting the known in vitro findings.

Residue-residue contacts: application to analysis of secondary structure interactions.

Protein structures and their complexes are formed and stabilized by interactions, both inside and outside of the protein. Analysis of such interactions helps in understanding different levels of structures (secondary, super-secondary, and oligomeric states). It can also assist molecular biologists in understanding structural consequences of modifying proteins and/or ligands. In this chapter, our definition of atom-atom and residue-residue contacts is described and applied to analysis of protein-protein interactions in dimeric ß-sandwich proteins.

First- and second-shell metal binding residues in human proteins are disproportionately associated with disease-related SNPs.

Protein structure serves as a key determinant for revealing the molecular basis of human disease. Metal ions are among the most frequently bound heterogroups in proteins affecting structure and function. We analyzed the relationship between single nucleotide polymorphisms (SNPs) associated with human disease and metal binding sites in proteins on a database scale, using structural models and predictive tools. A match was identified for 586 disease-associated SNPs (dSNPs) located at 135 predicted metal binding sites and associated with 126 diverse diseases. For 104 diseases, a metal is known to bind at the predicted site in the homologue; for 22, the analysis gives a first indication for metal involvement in the disease. As second-shell residues play an important part in metal ion binding, our analysis included protein space up to 4.5 Å from metal binding sites. The ratio of disease-associated versus nondisease-associated SNPs (dSNP/ndSNP) for first-shell residues is 7.4 and for second-shell residues, 3.1. In addition, over 13% of all dSNPs were found to be associated with first- and second-shell residues, although these residues occupy only about 3% of protein space. These results show a disproportionate association of dSNPs and metal binding sites over a wide variety of diseases.

Nitric oxide donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked.

An outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair cycle. D1 also undergoes redox-dependent phosphorylation prior to its degradation. Although it has been suggested that phosphorylation modulates D1 metabolism, reversible D1 phosphorylation was reported not to be essential for PS II repair in Arabidopsis. Thus, the involvement of phosphorylation in D1 degradation is controversial. We show here that nitric oxide donors inhibit in vivo phosphorylation of the D1 protein in Spirodela without inhibiting degradation of the protein. Thus, D1 phosphorylation is not tightly linked to D1 degradation in the intact plant.

Prediction of 3D metal binding sites from translated gene sequences based on remote-homology templates.

Database-scale analysis was performed to determine whether structural models, based on remote homologues, are effective in predicting 3D transition metal binding sites in proteins directly from translated gene sequences. The extent by which side chain modeling alone reduces sensitivity and selectivity is shown to be <10%. Surprisingly, selectivity was not dependent on the level of sequence homology between template and target, or on the presence of a metal ion in the structural template. Applying a modification of the CHED algorithm (Babor et al., Proteins 2008;70:208-217) and machine learning filters, a selectivity of approximately 90% was achieved for protein sequences using unrelated structural templates over a sequence identity range of 18-100%. Below approximately 18% identity, the number of analyzable target-template pairs and predictability of metal binding sites falls off sharply. A full third of structural templates were found to have target partners only in the remote homology range of 18-30%. In this range, nonmetal-binding templates are calculated to be the majority and serve to predict with 50% sensitivity at the geometric level. Overall, sensitivity at the geometric level for targets having templates in the 18-30% sequence identity range is 73%, with an average of one false positive site per true site. Protein sequences described as "unknown" in the UniProt database and composed largely of unidentified genome project sequences were studied and metal binding sites predicted. A web server for prediction of metal binding sites from protein sequence is provided.

D1-protein dynamics in photosystem II: the lingering enigma.

The D1/D2 heterodimer core is the heart of the photosystem II reaction center. A characteristic feature of this heterodimer is the differentially rapid, light-dependent degradation of the D1 protein. The D1 protein is possibly the most researched photosynthetic polypeptide, with aspects of structure-function, gene, messenger and protein regulation, electron transport, reactive oxygen species, photoinhibition, herbicide binding, stromal-granal translocations, reversible phosphorylation, and specific proteases, all under intensive investigation more than three decades after the protein's debut in the literature. This review will touch on some treaded areas of D1 research that have, so far, defied clear resolution, as well as cutting edge research on mechanisms and consequences of D1 protein degradation.

Prediction of transition metal-binding sites from apo protein structures.

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.

Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin.

Aprotinin is a small serine protease inhibitor used in human health. Spirodela were transformed, via Agrobacterium, with a synthetic gene encoding the mature aprotinin sequence and a signal peptide for secretion which was driven by the CaMV 35S promoter. A total of 25 transgenic Spirodela lines were generated and aprotinin production was confirmed by northern and western blot analyses. Expression levels of up to 3.7% of water soluble proteins were detected in the plant and 0.65 mg/l in the growth medium. In addition, immunoaffinity purification of the protein followed by amino acid sequencing confirmed the correct splicing of the aprotinin produced in Spirodela and secreted into the growth medium.

High expression of transgene protein in Spirodela.

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.

The limit of accuracy of protein modeling: influence of crystal packing on protein structure.

The size of the protein database (PDB) makes it now feasible to arrive at statistical conclusions regarding structural effects of crystal packing. These effects are relevant for setting upper practical limits of accuracy on protein modeling. Proteins whose crystals have more than one molecule in the asymmetric unit or whose structures were determined at least twice by X-ray crystallography were paired and their differences analyzed. We demonstrate a clear influence of crystal environment on protein structure, including backbone conformations, hinge-like motions and side-chain conformations. The positions of surface water molecules tend to be variable in different crystal environments while those of ligands are not. Structures determined by independent groups vary more than structures determined by the same authors. The use of different refinement methods is a major source for this effect. Our pair-wise analysis derives a practical limit to the accuracy of protein modeling. For different crystal forms, the limit of accuracy (C(alpha), root-mean-square deviation (RMSD)) is approximately 0.8A for the entire protein, which includes approximately 0.3A due to crystal packing. For organized secondary elements, the upper limit of C(alpha) RMSD is 0.5-0.6A while for loops or protein surface it reaches 1.0A. Twenty percent of exposed side- chains exhibit different chi(1+2) conformations with approximately half of the effect also resulting from crystal packing. A web based tool for analysis and graphic presentation of surface areas of crystal contacts is available (http://ligin.weizmann.ac.il/cryco).

SPACE: a suite of tools for protein structure prediction and analysis based on complementarity and environment.

We describe a suite of SPACE tools for analysis and prediction of structures of biomolecules and their complexes. LPC/CSU software provides a common definition of inter-atomic contacts and complementarity of contacting surfaces to analyze protein structure and complexes. In the current version of LPC/CSU, analyses of water molecules and nucleic acids have been added, together with improved and expanded visualization options using Chime or Java based Jmol. The SPACE suite includes servers and programs for: structural analysis of point mutations (MutaProt); side chain modeling based on surface complementarity (SCCOMP); building a crystal environment and analysis of crystal contacts (CryCo); construction and analysis of protein contact maps (CMA) and molecular docking software (LIGIN). The SPACE suite is accessed at http://ligin.weizmann.ac.il/space.

Flexibility of metal binding sites in proteins on a database scale.

Protein metal binding sites in the pre-bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo-holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side-chain rearrangement inversely correlates with the number of first-shell residues. Analysis of side-chain reorientations as a result of metal binding showed that in 95% of the rigid-backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre-bound form. The frequencies of side-chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state.