Juan Vorster

Neglecting legumes has compromised human health and sustainable food production

The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner ‘nutritious seeds for a sustainable future’. A second green revolution is required to ensure food and nutritional security in the face of global climate change. Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming. In addition, a legume-rich diet has health benefits for humans and livestock alike. However, grain legumes form only a minor part of most current human diets, and legume crops are greatly under-used. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. The current lack of coordinated focus on grain legumes has compromised human health, nutritional security and sustainable food production.

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg-MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin-infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.

Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities

Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

Screening of Tea (Camellia sinensis) for Trait-Associated Molecular Markers

This study was done to identify random amplified polymorphic DNA (RAPD) markers that may associate with seven important traits in tea. Sixty RAPD primers were first screened using 18 cultivars under each of the 7 traits, followed by confirmatory screening of 20 promising primers with 32 tea cultivars. Six RAPD primers generated a total of nine specific bands that associated with six desired traits: black tea quality and tolerance to drought, high temperature, low temperature, Phomopsis theae, and high yield. These markers would allow early identification of plant material with the desired traits that can be advanced to the next stage of selection and enhance targeted choice of breeding stocks with the desirable traits. The nine RAPD markers identified in this study could improve precision and efficiency in tea breeding and selection and are an important contribution towards the establishment of marker-assisted selection in tea breeding programmes.

A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

The use of multiplexed simple sequence repeat (SSR) markers for analysis of genetic diversity in African rice genotypes

Rice is an emerging food and cash crop in Eastern Africa. Thousands of germplasm accessions have been introduced from major rice breeding centers, such as the International Rice Research Institute (IRRI), and Africa Rice but the genetic variability among the introduced rice germplasm is unknown. Knowledge on genetic diversity would be useful in designing measures for comprehensive breeding and conservation. To address this knowledge gap, 10 highly polymorphic rice simple sequence repeat (SSR) markers were used to characterize 99 rice genotypes to determine their diversity and place them in their different population groups. The SSR markers were multiplexed in 3 panels to increase their throughput. An average of 15.9 alleles was detected, ranging from 6 alleles detected by marker RM7 to 30 by marker RM333. The UPGMA dendogram based on Nei’s genetic distance cluster analysis, revealed 5 genetic groups among the genotypes tested. Analysis of molecular variance indicated that 97% of the diversity observed was explained by differences in the genotypes themselves, and only 3% was due to the sources from which the genotypes were obtained. This study sets the stage for further diversity analysis of all the available germplasm lines using SSR markers to ensure effective utilization and conservation of the germplasm. Keywords: Genetic diversity, simple sequences repeat (SSR) markers, multiplexing, rice genotypes, structure. Abbreviation: IRRI, International Rice Research Institute; SSR, simple sequence repeat; NaCRRI, national crops resources research institute; RAPD, random amplified polymorphic DNA; AFLP, amplified fragment length polymorphisms; RFLP, restriction fragment length polymorphisms; SNP, single nucleotide polymorphisms; BAC, bacterial artificial chromosome; PAC, P1-derived artificial chromosome; PCR, polymerase chain reaction; Ho, heterozygosity; He, heterozygosity.

Microsatellite association with seed protein content and flowering time in Nigerian cowpea cultivars

Cowpea microsatellites were evaluated for their potential to be used as markers associated with seed protein content and plant flowering time. Seeds with high and low protein content as well as plants with early and late flowering time were selected from 104 Nigerian cowpea accessions. Fifteen (15) microsatellite primer pairs were tested for amplification of microsatellite sequences using PCR and seven pairs amplified a single-locus. Sequence analysis showed a high level of diversity between the analysed genotypes. Although, none of the microsatellites allowed clustering for protein content, one primer pair, VM68, detected a (AG)12-repeat microsatellite allele, which was present only in plants with late flowering. This microsatellite might possibly be useful as a marker associated with late-flowering time. Key words : Cowpea, microsatellite, protein content, flowering time.

Orphan Legumes Growing in Dry Environments: Marama Bean as a Case Study

Plants have developed morphological, physiological, biochemical, cellular, and molecular mechanisms to survive in drought-stricken environments with little or no water caused by below-average precipitation. In this mini-review, we highlight the characteristics that allows marama bean [Tylosema esculentum (Burchell) Schreiber], an example of an orphan legume native to arid regions of southwestern Southern Africa, to flourish under an inhospitable climate and dry soil conditions where no other agricultural crop competes in this agro-ecological zone. Orphan legumes are often better suited to withstand such harsh growth environments due to development of survival strategies using a combination of different traits and responses. Recent findings on questions on marama bean speciation, hybridization, population dynamics, and the evolutionary history of the bean and mechanisms by which the bean is able to extract and conserve water and nutrients from its environment as well as aspects of morphological and physiological adaptation will be reviewed. The importance of the soil microbiome and the genetic diversity in this species, and their interplay, as a reservoir for improvement will also be considered. In particular, the application of the newly established marama bean genome sequence will facilitate both the identification of important genes involved in the interaction with the soil microbiome and the identification of the diversity within the wild germplasm for genes involved drought tolerance. Since predicted future changes in climatic conditions, with less water availability for plant growth, will severely affect agricultural productivity, an understanding of the mechanisms of unique adaptations in marama bean to such conditions may also provide insights as to how to improve the performance of the major crops.