The complex response of free and bound amino acids to water stress during the seed setting stage in Arabidopsis

Free amino acids (FAAs) and protein‐bound amino acids (PBAAs) in seeds play an important role in seed desiccation, longevity, and germination. However, the effect that water stress has on these two functional pools, especially when imposed during the crucial seed setting stage is unclear. To better understand these effects, we exposed Arabidopsis plants at the seed setting stage to a range of water limitation and water deprivation conditions and then evaluated physiological, metabolic, and proteomic parameters, with special focus on FAAs and PBAAs. We found that in response to severe water limitation, seed yield decreased, while seed weight, FAA, and PBAA content per seed increased. Nevertheless, the composition of FAAs and PBAAs remained unaltered. In response to severe water deprivation, however, both seed yield and weight were reduced. In addition, major alterations were observed in both FAA and proteome compositions, which indicated that both osmotic adjustment and proteomic reprogramming occurred in these naturally desiccation‐tolerant organs. However, despite the major proteomic alteration, the PBAA composition did not change, suggesting that the proteomic reprogramming was followed by a proteomic rebalancing. Proteomic rebalancing has not been observed previously in response to stress, but its occurrence under stress strongly suggests its natural function. Together, our data show that the dry seed PBAA composition plays a key role in seed fitness and therefore is rigorously maintained even under severe water stress, while the FAA composition is more plastic and adaptable to changing environments, and that both functional pools are distinctly regulated.     

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.     

Coordinated gene networks regulating Arabidopsis plant metabolism in response to various stresses and nutritional cues

The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.     

Sexual Cannibalism in the Brown Widow Spider (Latrodectus geometricus)

Sexual cannibalism may represent an extreme form of male monogamy. According to this view, males gain reproductive success by sacrificing themselves to females. We studied the occurrence and timing of sexual cannibalism in the brown widow spider Latrodectus geometricus and compared male courtship and mating behavior with virgin and with previously mated females. We found that events of sexual cannibalism are frequent, that they occur during copulation and that males initiate cannibalism by placing the abdomen in front of the female’s mouth‐parts during copulation (somersault behavior). Both the somersaults and mating occurred more frequently with virgins than with previously mated females. Our results support the hypothesis that sexual cannibalism is a male strategy in this species. The somersault behavior was previously known only from the redback spider, Latrodectus hasselti. It is as yet unknown whether self‐sacrifice has evolved more than once in this genus.     

Landscape-scale regeneration dynamics of disturbed Mediterranean maquis

Abstract. Long-term regeneration dynamics of Mediterranean maquis was investigated by analysing historical aerial photographs of Mt. Carmel, one of the largest protected areas in the Mediterranean region of Israel. Two sets of aerial photographs were processed, one from 1960 (before the area was protected), and the second from 1992 (21 yr after the area was declared a nature reserve). The photographs of each year were classified into three vegetation states based on the percentage cover of trees: open maquis with tree cover < 33.3 %, moderately developed maquis (tree cover 33.3–66.6 %), and closed maquis (tree cover > 66.6 %). Grid maps constructed from the classified images were used to determine probabilities of transition between vegetation states. Closed maquis showed zero probability of transition to either open or moderately developed maquis. Probabilities of ‘forward’ transitions (transitions from low-cover to high-cover classes) were higher on north-facing than on south-facing slopes. On north-facing slopes, the area of open maquis decreased from 87 % to 46 % during the period studied, while that of closed maquis increased from 3 % to 29 %. On south-facing slopes open maquis decreased from 87 % to 69 % while closed maquis increased from 1 % to 8 %. Within a particular aspect, tree cover in 1960 was a reliable predictor of tree cover in 1992. This indicates that micro-scale patterns of tree distribution in 1960 were important in determining the structure of the maquis 32 yr later. Simulations based on the empirically derived transition probabilities suggest that, under current climatic conditions, the process of maquis regeneration on Mt. Carmel may take 500–1000 yr.     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative "hot spot" common to all scorpion beta-toxins affecting Na+ channels

Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.