Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Evidence for structural heterogeneity from molecular cytogenetic analysis of dicentric Robertsonian translocations.

Most Robertsonian translocations are dicentric, suggesting that the location of chromosomal breaks leading to their formation occur in the acrocentric short arm. Previous cytogenetic and molecular cytogenetic studies have shown that few Robertsonian translocations retain ribosomal genes or beta-satellite DNA. Breakpoints in satellite III DNA, specifically between two chromosome 14-specific subfamilies, pTRS-47 and pTRS-63, have been indicated for most of the dicentric 14q21q and 13q14q translocations that have been studied. We have analyzed the structure of 36 dicentric translocations, using several repetitive DNA probes that localize to the acrocentric short arm. The majority of the translocations retained satellite III DNA, while others proved variable in structure. Of 10 14q21q translocations analyzed, satellite III DNA was undetected in 1; 6 retained one satellite III DNA subfamily, pTRS-47; and 3 appeared to contain two 14-specific satellite III DNA sub-families, pTRS-47 and pTRS-63. In 10/11 translocations involving chromosome 15, the presence of satellite III DNA was observed. Our results show that various regions of the acrocentric short arm, and, particularly, satellite III DNA sequences, are involved in the formation of Robertsonian translocations.     

A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid Biosynthesis

Red clover (Trifolium pratense) leaves accumulate several μmol g⁻¹ fresh weight of phaselic acid [2-O-(caffeoyl)-l-malate]. Postharvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forage crops like alfalfa (Medicago sativa) lack both polyphenol oxidase and o-diphenols, and breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forage crops utilizing this natural system of protein protection. A possible pathway for phaselic acid biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs were identified in red clover. HCT1 shares more than 75% amino acid identity with a number of well-characterized shikimate O-HCTs implicated in monolignol biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has limited sequence identity to any previously identified HCT. Expression analyses indicate that HCT1 mRNA accumulates to 4-fold higher levels in stems than in leaves, whereas HCT2 mRNA accumulates to 10-fold higher levels in leaves than in stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate that HCT1 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to shikimate but not malate, whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to malate but not shikimate. Together, these results indicate that HCT1 is involved in monolignol biosynthesis and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.In contrast to many other forage legumes (e.g. alfalfa [Medicago sativa]; Jones et al., 1995), red clover (Trifolium pratense) accumulates relatively high levels of the phenylpropanoid o-diphenol phaselic acid [2-O-(caffeoyl)-l-malic acid; hereafter referred to as caffeoyl-malate or phaselic acid] in its leaves (Hatfield and Muck, 1999; Winters et al., 2008). In red clover, upon cellular disruption, phaselic acid and other o-diphenols are readily oxidized by a soluble polyphenol oxidase (PPO) to produce their corresponding o-quinones (Hatfield and Muck, 1999; Sullivan et al., 2004). The formation of such o-quinones by PPO, and the subsequent secondary reactions of these quinones, are most often associated with browning of fresh fruits and vegetables (Steffens et al., 1994), which has a negative impact on perceived quality. When preserved by ensiling, however, oxidation of o-diphenols by PPO in red clover prevents degradation of protein during storage (Sullivan et al., 2004; Sullivan and Hatfield, 2006). Although alfalfa lacks significant levels of both PPO activity and o-diphenol compounds in its leaves, red clover''s natural system of protein protection has been transferred to this forage legume by expressing a red clover PPO transgene in alfalfa and exogenously adding o-diphenol PPO substrates to the resulting tissues or tissue extracts (Sullivan et al., 2004; Sullivan and Hatfield, 2006). Because ruminant animals poorly utilize degraded protein, adaptation of the PPO system to alfalfa and other forage crops would have substantial positive economic and environmental impacts (Sullivan and Hatfield, 2006). Unfortunately, lack of system components in these forage crops, especially the o-diphenol PPO substrates, presents a challenge to practical adaptation of this natural system of protein preservation. Consequently, understanding how red clover is able to accumulate o-diphenols such as phaselic acid will be a key step to adapt the PPO/o-diphenol system to a wide range of economically important forage crops.The biosynthetic pathways whereby red clover synthesizes and accumulates phaselic acid and other o-diphenols have not been defined. However, in the Brassicaceae, hydroxycinnamoyl esters with malic acid can be made via the action of sinapoyl-Glc:malate sinapoyltransferase (SMT; EC 2.3.1), which is capable of transferring a hydroxycinnamoyl moiety from a hydroxycinnamoyl-Glc ester to a malic acid acceptor. In Arabidopsis (Arabidopsis thaliana), SNG1 (for sinapoylglucose accumulator 1), which encodes the enzyme, has been shown to be responsible for the accumulation of sinapoylmalate in seeds and leaves (Lehfeldt et al., 2000). An SMT from radish (Raphanus sativus), presumably the homolog of the Arabidopsis SNG1 gene product, has been purified to apparent homogeneity and characterized (Grawe et al., 1992). The purified enzyme is capable of utilizing sinapoyl-, feruloyl-, caffeoyl-, and to a lesser extent p-coumaroyl-Glc esters to form the corresponding malic acid esters, suggesting that it is responsible for the accumulation of these esters in vivo. In contrast, in many plants, formation of certain hydroxycinnamoyl esters is often mediated by a member of the BAHD transferase family (D''Auria, 2006) that utilize a CoA thiolester hydroxycinnamoyl donor. Some of the best characterized of these hydroxycinnamoyl transferases (HCTs) are those associated with the biosynthesis of monolignols (Hoffmann et al., 2003, 2004; Shadle et al., 2007). These are capable of transferring p-coumaroyl or caffeoyl moieties from the respective CoA thiolesters to form 5-O-esters with shikimic acid or, to a lesser extent, 3-O-esters with quinic acid. Separable enzymatic activities capable of transferring a p-coumaroyl moiety to either shikimate/quinate or to 4′-hydroxyphenyllactate in basil (Ocimum basilicum) peltate gland extracts have been identified, although genes encoding these activities have not been cloned (Gang et al., 2002). Niggeweg et al. (2004) used gene-silencing experiments to definitively demonstrate that a hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) is responsible for chlorogenic acid accumulation in the Solanaceae. Although phaselic acid biosynthesis in red clover could be via a pathway utilizing SMT, lack of an apparent SNG1 homolog in a collection of red clover EST sequences derived from leaves and young plants suggests otherwise (see “Discussion”). Therefore, pathways in red clover for the biosynthesis of phaselic acid utilizing one or more BAHD family transferase (Fig. 1) should be considered. In these proposed pathways, Phe would be converted to p-coumaroyl-CoA by the sequential action of Phe ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL). The action of one or more specific HCTs and one or more p-coumarate 3′-hydroxylases (C3Hs) would then result in the formation of phaselic acid.Open in a separate windowFigure 1.Possible pathways for phaselic acid biosynthesis in red clover. Proposed pathway enzymes for the production of phaselic acid include PAL, 4CL, hydroxycinnamoyl:shikimate transferase (HCT-S), hydroxycinnamoyl:malate transferase (HCT-M), and C3H. The branch point at p-coumaroyl-CoA represents two alternative pathways. For simplicity, not all reactants and products are shown.Existing literature suggests that C3H enzymes, which are cytochrome P450 enzymes (CYP98A subfamily), do not directly hydroxylate p-coumaric acid to caffeic acid but rather act on p-coumaroyl ester derivatives. For example, the enzyme from Arabidopsis hydroxylates shikimic and quinic acid esters of p-coumaric acid but only poorly or not at all p-coumaric acid or its Glc or CoA esters (Schoch et al., 2001; Franke et al., 2002). Thus, one model of phaselic acid biosynthesis is the formation of 2-O-(p-coumaroyl)-l-malic acid (hereafter referred to as p-coumaroyl-malate) by a HCT and its subsequent hydroxylation by a C3H enzyme capable of utilizing the malic acid ester as a substrate (Fig. 1, bottom, red pathway). An alternative model would require at least two HCT activities for phaselic acid biosynthesis (Fig. 1, top, blue pathway). The first activity would form a substrate suitable for hydroxylation (e.g. p-coumaroyl-shikimate, since several characterized C3H enzymes appear to favor this substrate [Schoch et al., 2001; Franke et al., 2002; Gang et al., 2002; Morant et al., 2007]). Following hydroxylation to the caffeoyl derivative by a C3H, the first HCT activity could synthesize caffeoyl-CoA via its reverse reaction (Hoffmann et al., 2003; Niggeweg et al., 2004). A second HCT activity would then transfer the caffeoyl moiety to malic acid to form phaselic acid. Both pathways predict a transferase capable of transferring a hydroxycinnamoyl moiety (either p-coumaroyl or caffeoyl) to malic acid. Also, these pathways are consistent with the observation that, at least in vitro, several characterized HCT enzymes are capable of transfer reactions utilizing either p-coumaroyl- or caffeoyl-CoA (Hoffmann et al., 2003; Niggeweg et al., 2004). The identification and characterization of two distinct HCTs from red clover, one of which has properties consistent with a role in phaselic acid biosynthesis, are reported here.     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Fitness consequences of pheromone production and host selection strategies in a tree-killing bark beetle (Coleoptera: Curculionidae: Scolytinae)

Timing of arrival at a resource often determines an individual’s reproductive success. Tree-killing bark beetles can reproduce in healthy trees by attacking in adequate numbers to overcome host defences that could otherwise be lethal. This process is mediated by aggregation and antiaggregation pheromones. Beetles that arrive early in such a “mass attack” must contend with undiminished tree defences, and produce enough pheromones to attract more beetles, but have a head start on gallery construction and egg-laying. Beetles that arrive late may be impeded by competition and diminishing availability of phloem, but should experience fewer costs associated with pheromone production and battling tree defences. We investigated relationships between timing of arrival, body size, pheromone production and fitness in the southern pine beetle, Dendroctonus frontalis. In field experiments, we captured beetles that arrived early (pioneers) and late on slash pine trees, Pinus elliottii, and measured pheromone amounts in their hindguts. We marked gallery entrances of beetles as they landed on a tree and measured their reproductive success after the attack terminated. We found no difference in body size or pheromone amounts between early and late arrivers. Most beetles arrived at the middle of the attack sequence, and excavated longer galleries per day than early arrivers. The number of offspring produced per day by beetles that established galleries midway through mass attack was higher than those that arrived early or very late in the sequence. Our results suggest that beetles do not exhibit adaptive phenotypic plasticity in pre-landing pheromone production, depending on the extent of previous colonisation of a host. Rather, it appears that stabilising selection favours beetles that attack in the middle of the sequence, and contributes to attack synchrony. Synchronous attack on trees is essential before population booms characteristic of tree-killing bark beetles can occur in nature.     

Photosynthetic capacity in microalgae associated with Antarctic pack ice

Summary Previous studies of primary production in Antarctic seas have concluded that microalgae associated with sea ice make only a minor contribution to the carbon budget; however, production estimates for sea ice algae have been based almost exclusively on microalgae from nearshore fast ice. We measured biomass and rates of photosynthesis (at saturating irradiances) in microalgae collected from offshore pack ice during four cruises to the Weddell-Scotia Sea and the region west of the Antarctic Peninsula. Chlorophyll a concentrations in pack ice (0.089 to 260 g 1^-1) were as high as reported from fast ice. Photosynthetic rates typically ranged (median 75%) from 0.3 to 3.6 C g chl a ^-1 h^-1 (n=127; arithmetic mean = 1.7, S D =1.9). These photosynthetic capacities are approximately an order of magnitude greater than previously reported for fast ice microalgae, but are similar to rates reported for Antarctic phytoplankton. Because pack ice constitutes more than 90% of the ice cover in Antarctic seas and indigenous microalgae have a higher photosynthetic capacity than previously realized, we raise the question: has the importance of sea ice algae to primary product: on in the southern ocean been underestimated?