Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>The complex responses of plants to DNA damage are incompletely understood and the role of members of the serpin protein family has not been investigated. Serpins are functionally diverse but structurally conserved proteins found in all three domains of life. In animals, most serpins have regulatory functions through potent, irreversible inhibition of specific serine or cysteine proteinases via a unique suicide-substrate mechanism. Plant serpins are also potent proteinase inhibitors, but their physiological roles are largely unknown.</p> <p>Results</p> <p>Six <it>Arabidopsis </it>genes encoding full-length serpins were differentially expressed in developing seedlings and mature tissues. Basal levels of <it>AtSRP2 </it>(At2g14540) and <it>AtSRP3 </it>(At1g64030) transcripts were highest in reproductive tissues. <it>AtSRP2 </it>was induced 5-fold and <it>AtSRP3 </it>100-fold after exposure of seedlings to low concentrations of methyl methanesulfonate (MMS), a model alkylating reagent that causes DNA damage. Homozygous T-DNA insertion mutants <it>atsrp2 </it>and <it>atsrp3 </it>exhibited no differential growth when mutant and wild-type plants were left untreated or exposed to γ-radiation or ultraviolet light. In contrast, <it>atsrp2 </it>and <it>atsrp3 </it>plants exhibited greater root length, leaf number and overall size than wild-type plants when exposed to MMS. Neither of the two serpins was required for meiosis. GFP-AtSRP2 was localized to the nucleus, whereas GFP-AtSRP3 was cytosolic, suggesting that they target different proteinases. Induction of cell cycle- and DNA damage-related genes <it>AtBRCA1</it>, <it>AtBARD1</it>, <it>AtRAD51</it>, <it>AtCYCB1;1 </it>and <it>AtCYCD1;1</it>, but not <it>AtATM</it>, was reduced relative to wild-type in <it>atsrp2 </it>and <it>atsrp3 </it>mutants exposed to MMS.</p> <p>Conclusion</p> <p>Expression of specific serpin genes (<it>AtSRP2 </it>and <it>AtSRP3 </it>in <it>Arabidopsis</it>) is required for normal responses of plants following exposure to alkylating genotoxins such as MMS.</p

Computationally Designed Bispecific Antibodies using Negative State Repertoires

A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies

Heat tolerance in a wild Oryza species is attributed to maintenance of Rubisco activation by a thermally stable Rubisco activase ortholog

• The response of photosynthesis and plant growth to short periods of supra-optimal heat was tested in rice (Oryza sativa) and two wild Oryza species from the Australian savanna, O. meridionalis and O. australiensis. The mechanism of heat tolerance in the wild species was explored, particularly focusing on the heat-labile protein Rubisco activase (RCA). • We compared leaf elongation rates, net photosynthesis and Rubisco activation state at moderate (28°C) and high temperature (45°C). Sequence analysis followed by enzyme kinetics of RCA was used to identify structural differences and thermal stability. • Oryza australiensis was the most heat-tolerant species. Rubisco activation state was positively correlated with leaf elongation rates across all three species at four times following exposure to 45°C. Oryza australiensis had multiple polymorphisms in the RCA primary protein sequence, and the protein was thermally stable up to 42°C relative to RCA from O. sativa which became inhibited at 36°C. • We attribute the heat tolerance of growth and photosynthesis in these wild species to thermal stability of RCA, enabling Rubisco to remain active. Because thermal stability of RCA in O. australiensis co-occurs with reduced enzyme specific activity, an increased RCA to Rubisco ratio is required in vivo to maintain high Rubisco activation

Community recommendations on terminology and procedures used in flooding and low oxygen stress research

Apart from playing a key role in important biochemical reactions, molecular oxygen (O2) and its by-products also have crucial signaling roles in shaping plant developmental programs and environmental responses. Even under normal conditions, sharp O2 gradients can occur within the plant when cellular O2 demand exceeds supply, especially in dense organs such as tubers, seeds and fruits. Spatial and temporal variations in O2 concentrations are important cues for plants to modulate development (van Dongen & Licausi, 2015; Considine et al., 2016). Environmental conditions can also expand the low O2 regions within the plant. For example, excessive rainfall can lead to partial or complete plant submergence resulting in O2 deficiency in the root or the entire plant (Voesenek & Bailey-Serres, 2015). Climate change-associated increases in precipitation events have made flooding a major abiotic stress threatening crop production and food sustainability. This increased flooding and associated crop losses highlight the urgency of understanding plant flooding responses and tolerance mechanisms. Timely manifestation of physiological and morphological changes triggering developmental adjustments or flooding survival strategies requires accurate sensing of O2 levels. Despite progress in understanding how plants sense and respond to changes in intracellular O2 concentrations (van Dongen & Licausi, 2015), several questions remain unanswered due to a lack of high resolution tools to accurately and noninvasively monitor (sub)cellular O2 concentrations. In the absence of such tools, it is therefore critical for researchers in the field to be aware of how experimental conditions can influence plant O2 levels, and thus on the importance of accurately reporting specific experimental details. This also requires a consensus on the definition of frequently used terms. At the 15th New Phytologist Workshop on Flooding stress (Voesenek et al., 2016), community members discussed and agreed on unified nomenclature and standard norms for low O2 and flooding stress research. This consensus on terminology and experimental guidelines is presented here. We expect that these norms will facilitate more effective interpretation, comparison and reproducibility of research in this field. We also highlight the current challenges in noninvasively monitoring and measuring O2 concentrations in plant cells, outlining the technologies currently available, their strengths and drawbacks, and their suitability for use in flooding and low O2 research

Global Analysis of Arabidopsis/Downy Mildew Interactions Reveals Prevalence of Incomplete Resistance and Rapid Evolution of Pathogen Recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously

Well-designed experiments make proteomic studies on stressed plants meaningful

Analysis of the impact of abiotic stresses on plants is technically demanding. The cultivation of plants, application of treatments, choice of tissues and preparation of biological samples for proteomic analysis is as important as the subse’uent identification of proteins. With appropriate precautions, proteomics will greatly improve our understanding of the mechanisms of abiotic stress tolerance. Hence, this chapter summarises some of the major design faults that can compromise the interpretation of ‘stress experiments’. The examples of salt, drought, thermal stress and waterlogging are taken as representative of commonly encountered stresses, with recommendations for ways to avoid artefacts in design. The importance of interactions between these stresses is then discussed, pointing out the relevance of carefully constructed time courses and attendant physiological measurements to define the degree of stress. Tissue selection is also emphasised, recognising that stresses have differential impacts on different organs. Finally, the significance of choice of plant species is discussed, with recognition of the value of model species and the importance of expanding the range of taxa used if the full range of stress acclimation responses is to be identified through proteomics.18 page(s

Endogenous Ethylene Concentration Is Not a Major Determinant of Fruit Abscission in Heat-Stressed Cotton (Gossypium hirsutum L.)

We investigated the role of ethylene in the response of cotton to high temperature using cotton genotypes with genetically interrupted ethylene metabolism. In the first experiment, Sicot 71BRF and 5B (a lintless variant with compromised ethylene metabolism) were exposed to 45°C, either by instantaneous heat shock or by ramping temperatures by 3°C daily for 1 week. One day prior to the start of heat treatment, half the plants were sprayed with 0.8 mM of the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG). In a subsequent experiment, Sicot 71BRF and a putatively heat-tolerant line, CIM 448, were exposed to 36 or 45°C for 1 week, and half the plants were sprayed with 20 μM of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, (ACC). High temperature exposure of plants in both experiments was performed at the peak reproductive phase (65–68 days after sowing). Elevated temperature (heat shock or ramping to 45°C) significantly reduced production and retention of fruits in all cotton lines used in this study. At the termination of heat treatment, cotton plants exposed to 45°C had at least 50% fewer fruits than plants under optimum temperature in all three genotypes, while plants at 36°C remained unaffected. Heat-stressed plants continued producing new squares (fruiting buds) after termination of heat stress but these squares did not turn into cotton bolls due to pollen infertility. In vitro inhibition of pollen germination by high temperatures supported this observation. Leaf photosynthesis (Pn) of heat-stressed plants (45°C) measured at the end of heat treatments remained significantly inhibited, despite an increased leaf stomatal conductance (gs), suggesting that high temperature impairs Pn independently of stomatal behavior. Metabolic injury was supported by high relative cellular injury and low photosystem II yield of the heat-stressed plants, indicating that high temperature impaired photosynthetic electron transport. Both heat shock and ramping of heat significantly reduced ethylene release from cotton leaf tissues measured at the end of heat treatment but modulating ethylene production via AVG or ACC application had no significant effect on fruit production or retention in heat-stressed cotton plants. Instead, high temperature accelerated fruit abortion by impairing pollen development and/or restricting leaf photosynthesis

Does soil nitrogen influence growth, water transport and survival of snow gum (Eucalyptus pauciflora Sieber ex Sprengel.) under CO2 enrichment?

Eucalyptus pauciflora Sieber ex Sprengel. (snow gum) was grown under ambient (370 L-1) and elevated (700 L-1) atmospheric CO2 in open-top chambers (OTCs) in the field and temperature- controlled glasshouses. Nitrogen applications to the soil ranged from 0.1 to 2.75 g N per plant. Trees in the field at high N levels grew rapidly during summer, particularly in CO2-enriched atmosphere, but suffered high mortality during summer heatwaves. Generally, wider and more numerous secondary xylem vessels at the root-shoot junction in CO2-enriched trees conferred fourfold higher below-ground hydraulic conductance. Enhanced hydraulic capacity was typical of plants at elevated CO2 (in which root and shoot growth was accelerated), but did not result from high N supply. However, because high rates of N application consistently made trees prone to dehydration during heatwaves, glasshouse studies were required to identify the effect of N nutrition on root development and hydraulics. While the effects of elevated CO2 were again predominantly on hydraulic conductivity, N nutrition acted specifically by constraining deep root penetration into soil. Specifically, 15-40% shallower root systems supported marginally larger shoot canopies. Independent changes to hydraulics and root penetration have implications for survival of fertilized trees under elevated atmospheric CO 2, particularly during water stress