Mrinal Bhave

Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses

The hardness or texture of cereal grains is a primary determinant of their technological and processing quality. Among members of the Triticeae, most notably wheat, much of the variation in texture is controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. Puroindolines confer the three major texture classes of soft and hard common wheat and the very hard durum wheat. The protein products of these genes interact with lipids and are associated with the surface of isolated starch (as a protein fraction known as ‘friabilin’). During the past ten years a great diversity of alleles of both Puroindoline genes have been discovered and significant advances made in understanding the relationship between the gene presence/absence, sequence polymorphism and texture of cereal grains. Efforts have also focussed on Puroindoline and Gsp-1 genes in diploid progenitors, other Triticeae grasses and synthetic wheats in order to understand the evolution of this gene family and find potentially useful variants. The puroindoline homologues in other cereals such as rye and barley are also receiving attention. This work summarises new developments in molecular genetics of puroindolines in wheat and related Triticeae grasses, and the related genes in other cereals.

Methods used for the detection and subtyping of Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen responsible for non-invasive and invasive diseases in the elderly, pregnant women, neonates and immunocompromised populations. This bacterium has many similarities with other non-pathogenic Listeria species which makes its detection from food and environmental samples challenging. Subtyping of L. monocytogenes strains can prove to be crucial in epidemiological investigations, source tracking contamination from food processing plants and determining evolutionary relationships between different strains. In recent years there has been a shift towards the use of molecular subtyping. This has led to the development of new subtyping techniques such as multi-locus variable number tandem repeat analysis (MLVA) and multi-locus sequence based typing (MLST). This review focuses on the available methods for Listeria detection including immuno-based techniques and the more recently developed molecular methods and analytical techniques such as matrix-assisted laser desorption/ionisation time-of-flight based mass spectrometry (MALDI-TOF MS). It also includes a comparison and critical analysis of the available phenotypic and genotypic subtyping techniques that have been investigated for L. monocytogenes.

Nuclear Activity of ROXY1, a Glutaredoxin Interacting with TGA Factors, Is Required for Petal Development in Arabidopsis thaliana

Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs.

Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype

The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant–water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry.

Plant aquaporins with non-aqua functions: deciphering the signature sequences

Research in recent years on plant Major Intrinsic Proteins (MIPs), commonly referred to as ‘aquaporins’, has seen a vast expansion in the substrates found to be transported via these membrane channels. The diversity in sizes, chemical nature and physiological significance of these substrates has meant a need to critically analyse the possible structural and biochemical properties of MIPs that transport these, in order to understand their roles. In this work we have undertaken a comprehensive analysis of all plant MIPs, coming from different families, that have been proven to transport ammonia, boron, carbon dioxide, hydrogen peroxide, silicon and urea. The sequences were analysed for all primary selectivity-related motifs (NPA motifs, ar/R filter, P1–P5 residues). In addition, the putative regulatory phosphorylation and glycosylation sites and mechanistic regulators such as loop lengths have been analysed. Further, nine specificity-determining positions (SDPs) were predicted for each group. The results show the ar/R filter residues, P2–P4 positions and some of the SDPs are characteristic for certain groups, and O-glycosylation sites are unique to a subgroup while N-glycosylation was characteristic of the other MIPs. Certain residues, especially in loop C, and structural parameters such as loop lengths also contribute to the uniqueness of groups. The comprehensive analysis makes significant inroads into appraising the intriguing diversity of plant MIPs and their roles in fundamental life processes, and provides tools for plant selections, protein engineering and transgenics.

The PIP and TIP aquaporins in wheat form a large and diverse family with unique gene structures and functionally important features.

Aquaporins, members of major intrinsic proteins (MIPs), transport water across cellular membranes and play vital roles in all organisms. Adversities such as drought, salinity, or chilling affect water uptake and transport, and numerous plant MIPs are reported to be differentially regulated under such stresses. However, MIP genes have been not yet been characterized in wheat, the largest cereal crop. We have identified 24 PIP and 11 TIP aquaporin genes from wheat by gene isolation and database searches. They vary extensively in lengths, numbers, and sequences of exons and introns, and sequences and cellular locations of predicted proteins, but the intron positions (if present) are characteristic. The putative PIP proteins show a high degree of conservation of signature sequences or residues for membrane integration, water transport, and regulation. The TIPs are more diverse, some with potential for water transport and others with various selectivity filters including a new combination. Most genes appear to be expressed as expressed sequence tags, while two are likely pseudogenes. Many of the genes are highly identical to rice but some are unique, and many correspond to genes that show differential expression under salinity and/or drought. The results provide extensive information for functional studies and developing markers for stress tolerance.

Detection of Listeria monocytogenes from selective enrichment broth using MALDI-TOF mass spectrometry

UNLABELLED Conventional methods used for primary detection of Listeria monocytogenes from foods and subsequent confirmation of presumptive positive samples involve prolonged incubation and biochemical testing which generally require four to five days to obtain a result. In the current study, a simple and rapid proteomics-based MALDI-TOF MS approach was developed to detect L. monocytogenes directly from selective enrichment broths. Milk samples spiked with single species and multiple species cultures were incubated in a selective enrichment broth for 24h, followed by an additional 6h secondary enrichment. As few as 1 colony-forming unit (cfu) of L. monocytogenes per mL of initial selective broth culture could be detected within 30h. On applying the same approach to solid foods previously implicated in listeriosis, namely chicken pâté, cantaloupe and Camembert cheese, detection was achieved within the same time interval at inoculation levels of 10cfu/mL. Unlike the routine application of MALDI-TOF MS for identification of bacteria from solid media, this study proposes a cost-effective and time-saving detection scheme for direct identification of L. monocytogenes from broth cultures.This article is part of a Special Issue entitled: Trends in Microbial Proteomics. BIOLOGICAL SIGNIFICANCE Globally, foodborne diseases are major causes of illness and fatalities in humans. Hence, there is a continual need for reliable and rapid means for pathogen detection from food samples. Recent applications of MALDI-TOF MS for diagnostic microbiology focused on detection of microbes from clinical specimens. However, the current study has emphasized its use as a tool for detecting the major foodborne pathogen, Listeria monocytogenes, directly from selective enrichment broths. This proof-of-concept study proposes a detection scheme that is more rapid and simple compared to conventional methods of Listeria detection. Very low levels of the pathogen could be identified from different food samples post-enrichment in selective enrichment broths. Use of this scheme will facilitate rapid and cost-effective testing for this important foodborne pathogen.

Origin and diversification of land plant CC-type glutaredoxins.

Glutaredoxins (GRXs) are ubiquitous glutathione-dependent oxidoreductase enzymes implicated in redox homeostasis, particularly oxidative stress response. Three major classes of GRX genes exist, the CPYC, CGFS classes are present in all pro- and eukaryote species, whereas the CC-type class GRXs are specific to land plants. In the basal land plant Physcomitrella patens, only two CC-type GRXs are present, compared with 21 in Arabidopsis. In contrast, sizes of the CPYC and CGFS classes remained rather similar throughout plant evolution, raising the interesting question as to when the CC-type GRXs first originated and how and why they expanded during land plant evolution. Recent evidence suggests that CC-type GRXs may have been recruited during evolution into diverse plant-specific functions of flower development (ROXY1, ROXY2) and pathogenesis response (ROXY19/GRX480). In the present study, GRX genes from the genomes of a range of green algae and evolutionarily diverse land plant species were identified; Ostreococcus, Micromonas, Volvox, Selaginella, Vitis, Sorghum, and Brachypodium. Previously identified sequences from Chlamydomonas, Physcomitrella, Oryza, Arabidopsis, and Populus were integrated to generate a more comprehensive understanding of the forces behind the evolution of various GRX classes. The analysis indicates that the CC-type GRXs probably arose by diversification from the CPYC class, at a time coinciding with colonization of land by plants. This strong differential expansion of the CC-type class occurred exclusively in the angiosperms, mainly through paleopolyploidy duplication events shortly after the monocot–eudicot split, and more recently through multiple tandem duplications that occurred independently in five investigated angiosperm lineages. The presented data suggest that following duplications, subfunctionalization, and subsequent neofunctionalization likely facilitated the sequestration of land plant-specific CC-type GRXs into novel functions such as development and pathogenesis response.

Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry

Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and comparable discriminatory indices of 0.89 and 0.86, respectively. MALDI-TOF MS thus represents a rapid and cost-effective source-tracking technique for L. monocytogenes.

Genome-wide analysis of genes encoding FK506-binding proteins in rice

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl cis/trans isomerase enzymes, some of which can also operate as molecular chaperones. FKBPs comprise a large ubiquitous family, found in virtually every part of the cell and involved in diverse processes from protein folding to stress response. Higher plant genomes typically encode about 20 FKBPs, half of these found in the chloroplast thylakoid lumen. Several FKBPs in plants are regulators of hormone signalling pathways, with important roles in seed germination, plant growth and stress response. Some FKBP isoforms exists as homologous duplicates operating in finely tuned mechanisms to cope with abiotic stress. In order to understand the roles of the plant FKBPs, especially in view of the warming environment, we have identified and analysed the gene families encoding these proteins in rice using computational approaches. The work has led to identification of all FKBPs from the rice genome, including novel high molecular weight forms. The rice FKBP family appears to have evolved by duplications of FKBP genes, which may be a strategy for increased stress tolerance.