Two calcium-dependent protein kinases enhance maize drought tolerance by activating anion channel ZmSLAC1 in guard cells

Stomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S-type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S-type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca²⁺-dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought-sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA-activated S-type anion currents in maize guard cells, while the S-type anion currents were increased in the guard cells of ZmCPK35- and ZmCPK37-overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1-mediated Cl^- and NO₃^- currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.     

Exploring emergent properties in cellular homeostasis using OnGuard to model K and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell.     

The Structure of the Small Laccase from Streptomyces coelicolor Reveals a Link between Laccases and Nitrite Reductases

The X-ray structure of the two-domain laccase (small laccase) from Streptomyces coelicolor A3(2) was solved at 2.7-Å resolution. The enzyme differs significantly from all laccases studied structurally so far. It consists of two domains and forms trimers and hence resembles the quaternary structure of nitrite reductases or ceruloplasmins more than that of large laccases. There are three trinuclear copper clusters in the enzyme localized between domains 1 and 2 of each pair of neighbor chains. In this way, a similar geometry of the active site as seen in large laccases is ensured, albeit by different arrangements of domains and protein chains. Three copper ions of type 1 lie close to one another near the surface of the central part of the trimer, and, effectively, a trimeric substrate binding site is formed in their vicinity.     

A Chimeric Fusion Protein Engineered with Disparate Functionalities—Enzymatic Activity and Self-assembly

The fusion of protein domains is an important mechanism in molecular evolution and a valuable strategy for protein engineering. We are interested in creating fusion proteins containing both globular and structural domains so that the final chimeric protein can be utilized to create novel bioactive biomaterials. Interactions between fused domains can be desirable in some fusion protein applications, but in this case the optimal configuration will enable the bioactivity to be unaffected by the structural cross-linking. To explore this concept, we have created a fusion consisting of a thermostable aldo-keto reductase, two α-helical leucine zipper domains, and a randomly coiled domain. The resulting protein is bifunctional in that (1) it can self-assemble into a hydrogel material as the terminal leucine zipper domains form interprotein coiled-coil cross-links, and (2) it expresses alcohol dehydrogenase and aldo-keto reductase activity native to AdhD from Pyrococcus furiosus. The kinetic parameters of the enzyme are minimally affected by the addition of the helical appendages, and rheological studies demonstrate that a supramolecular assembly of the bifunctional protein building blocks forms a hydrogel. An active hydrogel is produced at temperatures up to 60 °C, and we demonstrate the functionality of the biomaterial by monitoring the oxidation and reduction of the native substrates by the gel. The design of chimeric fusion proteins with both globular and structural domains is an important advancement for the creation of bioactive biomaterials for biotechnology applications such as tissue engineering, bioelectrocatalysis, and biosensing and for the study of native assembled enzyme structures and clustered enzyme systems such as metabolons.     

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.     

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.     

Molecular evolution and diversity of dimeric alpha-amylase inhibitor gene in Kengyilia species (Triticeae: Poaceae)

Kengyilia Yen et J. L. Yang is a group of allohexaploid species with StYP genomic constitutions in the wheat tribe. To investigate the evolution and diversity of dimeric alpha-amylase inhibitor genes in the Kengyilia, forty-five homoeologous DAAI gene sequences were isolated from sampled Kengyilia species and analyzed together with those of its close relatives. These results suggested that (1) Kengyilia species from Central Asia and the Qinghai–Tibetan Plateau had different origins from those of the geographically differentiated P genome; (2) the St and P genomes of Kengyilia were donated by Pseudoroegneria and Agropyron, respectively, and the Y genome had an independent origin and showed an affinity with the St genome; (3) purifying selection dominated the DAAI gene members and the St-DAAI gene was evolving at faster rate than the P- and Y-DAAI genes in Kengyilia; and (4) natural selection was the main factor on the codon usage pattern of the DAAI gene in Kengyilia.