Comparative Analysis of Tylosema esculentum Mitochondrial DNA Revealed Two Distinct Genome Structures.

Tylosema esculentum, commonly known as the marama bean, is an underutilized legume with nutritious seeds, holding potential to enhance food security in southern Africa due to its resilience to prolonged drought and heat. To promote the selection of this agronomically valuable germplasm, this study assembled and compared the mitogenomes of 84 marama individuals, identifying variations in genome structure, single-nucleotide polymorphisms (SNPs), insertions/deletions (indels), heteroplasmy, and horizontal transfer. Two distinct germplasms were identified, and a novel mitogenome structure consisting of three circular molecules and one long linear chromosome was discovered. The structural variation led to an increased copy number of specific genes, nad5, nad9, rrnS, rrn5, trnC, and trnfM. The two mitogenomes also exhibited differences at 230 loci, with only one notable nonsynonymous substitution in the matR gene. Heteroplasmy was concentrated at certain loci on chromosome LS1 (OK638188). Moreover, the marama mitogenome contained an over 9 kb insertion of cpDNA, originating from chloroplast genomes, but had accumulated mutations and lost gene functionality. The evolutionary and comparative genomics analysis indicated that mitogenome divergence in marama might not be solely constrained by geographical factors. Additionally, marama, as a member from the Cercidoideae subfamily, tends to possess a more complete set of mitochondrial genes than Faboideae legumes.

Comparative analysis of 84 chloroplast genomes of Tylosema esculentum reveals two distinct cytotypes.

Tylosema esculentum (marama bean) is an important orphan legume from southern Africa that has long been considered to have the potential to be domesticated as a crop. The chloroplast genomes of 84 marama samples collected from various geographical locations in Namibia and Pretoria were compared in this study. The cp genomes were analyzed for diversity, including SNPs, indels, structural alterations, and heteroplasmy. The marama cp genomes ranged in length from 161,537 bp to 161,580 bp and contained the same sets of genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The genes rpoC2 and rpoB, and the intergenic spacers trnT-trnL and ndhG-ndhI were found to be more diverse than other regions of the marama plastome. 15 haplotypes were found to be divided into two groups, differing at 122 loci and at a 230 bp inversion. One type appears to have greater variability within the major genome present, and variations amongst individuals with this type of chloroplast genome seems to be distributed within specific geographic regions but with very limited sampling for some regions. However, deep sequencing has identified that within most of the individuals, both types of chloroplast genomes are present, albeit one is generally at a very low frequency. The inheritance of this complex of chloroplast genomes appears to be fairly constant, providing a conundrum of how the two genomes co-exist and are propagated through generations. The possible consequences for adaptation to the harsh environment in which T. esculentum survives are considered. The results pave the way for marama variety identification, as well as for understanding the origin and evolution of the bean.

The Multipartite Mitochondrial Genome of Marama (Tylosema esculentum).

Tylosema esculentum (marama bean), a wild legume from tropical Africa, has long been considered as a potential crop for local farmers due to its rich nutritional value. Genomics research of marama is indispensable for the domestication and varietal improvement of the bean. The chloroplast genome of marama has been sequenced and assembled previously using a hybrid approach based on both Illumina and PacBio data. In this study, a similar method was used to assemble the mitochondrial genome of marama. The mitochondrial genome of the experimental individual has been confirmed to have two large circles OK638188 and OK638189, which do not recombine according to the data. However, they may be able to restructure into five smaller circles through recombination on the 4 pairs of long repeats (>1 kb). The total length of marama mitogenome is 399,572 bp. A 9,798 bp DNA fragment has been found that is homologous to the chloroplast genome of marama, accounting for 2.5% of the mitogenome. In the Fabaceae family, the mitogenome of Millettia pinnata is highly similar to marama, including for both the genes present and the total size. Some genes including cox2, rpl10, rps1, and sdh4 have been lost during the evolution of angiosperms and are absent in the mitogenomes of some legumes. However, these remain intact and functional in marama. Another set of genes, rpl2, rps2, rps7, rps11, rps13, and rps19 are either absent, or present as pseudogenes, in the mitogenome of marama.

EMS Derived Wheat Mutant BIG8-1 (Triticum aestivum L.)-A New Drought Tolerant Mutant Wheat Line.

Drought response in wheat is considered a highly complex process, since it is a multigenic trait; nevertheless, breeding programs are continuously searching for new wheat varieties with characteristics for drought tolerance. In a previous study, we demonstrated the effectiveness of a mutant known as RYNO3936 that could survive 14 days without water. In this study, we reveal another mutant known as BIG8-1 that can endure severe water deficit stress (21 days without water) with superior drought response characteristics. Phenotypically, the mutant plants had broader leaves, including a densely packed fibrous root architecture that was not visible in the WT parent plants. During mild (day 7) drought stress, the mutant could maintain its relative water content, chlorophyll content, maximum quantum yield of PSII (Fv/Fm) and stomatal conductance, with no phenotypic symptoms such as wilting or senescence despite a decrease in soil moisture content. It was only during moderate (day 14) and severe (day 21) water deficit stress that a decline in those variables was evident. Furthermore, the mutant plants also displayed a unique preservation of metabolic activity, which was confirmed by assessing the accumulation of free amino acids and increase of antioxidative enzymes (peroxidases and glutathione S-transferase). Proteome reshuffling was also observed, allowing slow degradation of essential proteins such as RuBisCO during water deficit stress. The LC-MS/MS data revealed a high abundance of proteins involved in energy and photosynthesis under well-watered conditions, particularly Serpin-Z2A and Z2B, SGT1 and Calnexin-like protein. However, after 21 days of water stress, the mutants expressed ABC transporter permeases and xylanase inhibitor protein, which are involved in the transport of amino acids and protecting cells, respectively. This study characterizes a new mutant BIG8-1 with drought-tolerant characteristics suited for breeding programs.

Wheat Line "RYNO3936" Is Associated With Delayed Water Stress-Induced Leaf Senescence and Rapid Water-Deficit Stress Recovery.

Random mutagenesis was applied to produce a new wheat mutant (RYNO3926) with superior characteristics regarding tolerance to water deficit stress induced at late booting stage. The mutant also displays rapid recovery from water stress conditions. Under water stress conditions mutant plants reached maturity faster and produced more seeds than its wild type wheat progenitor. Wild-type Tugela DN plants died within 7 days after induction of water stress induced at late booting stage, while mutant plants survived by maintaining a higher relative moisture content (RMC), increased total chlorophyll, and a higher photosynthesis rate and stomatal conductance. Analysis of the proteome of mutant plants revealed that they better regulate post-translational modification (SUMOylation) and have increased expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) proteins. Mutant plants also expressed unique proteins associated with dehydration tolerance including abscisic stress-ripening protein, cold induced protein, cold-responsive protein, dehydrin, Group 3 late embryogenesis, and a lipoprotein (LAlv9) belonging to the family of lipocalins. Overall, our results suggest that our new mutant RYNO3936 has a potential for inclusion in future breeding programs to improve drought tolerance under dryland conditions.

Evaluation of the effects of sugarcane processing on the presence of GM DNA and protein in sugar.

The Brazilian Sucro-energy Sector produces both energy, in the form of ethanol fuel, industrial steam and electricity, and sugar. Centro de Tecnologia Canavieira (CTC), the leading Brazilian sugarcane breeding company, has developed a pipeline of insect-protected sugarcane varieties to control sugarcane borer damage. The goal of this manuscript is to present the results of studies with three genetically modified (GM) sugarcane varieties and to evaluate the published literature regarding the possible presence of GM sugarcane DNA or protein in raw or refined sugar. Specifically, two varieties of approved GM sugarcane, CTC91087-6 and CTC175-A, and an experimental CTC variety, were grown in four individual plots to produce four batches each of processed raw sugar using standard smaller-scale laboratory processing methods resulting in a total of 12 independent batches of raw sugar. Herein, we report the development of event-specific probes and DNA detection methods, designed to detect the junction of sugarcane genomic DNA and the inserted DNA of the two approved GM varieties. An identical approach was used for the testing of sugar made from the experimental CTC variety. The methodology used TaqMan® real-time PCR and ELISA assays validated for the four GM proteins expressed by these three events (Cry1Ab, Cry1Ac, NPTII, and PAT (bar)). The developed assays had very low limits of detection (LODs) for the various event-specific DNA probes (7.2-25 ng/g sugar) and insecticidal and selectable marker proteins (2.9-10.9 ng/g sugar). No event-specific DNA and no GM proteins were detectable in the 12 independent batches of raw sugar produced from these three GM sugarcane events. The results of this study, using very sensitive methods and testing several sugar batches, extend the conclusions of previous studies, reviewed herein, that showed the extensive degradation and removal of DNA and protein during sugarcane processing. Overall, these results indicate that there are no distinguishable differences between the highly purified, chemically defined sugar produced from conventional or GM varieties.

Plant Vacuolar Processing Enzymes.

Plant proteomes contain hundreds of proteases divided into different families based on evolutionary and functional relationship. In particular, plant cysteine proteases of the C1 (papain-like) and C13 (legumain-like) families play key roles in many physiological processes. The legumain-like proteases, also called vacuolar processing enzymes (VPEs), perform a multifunctional role in different plant organs and during different stages of plant development and death. VPEs are similar to animal caspases, and although caspase activity was identified in plants almost 40 years ago, there still remains much research to be done to gain a complete understanding of their various roles and functions in plants. Here we not only summarize the current existing knowledge of plant VPEs, including recent developments in the field, but also highlight the future prospective areas to be investigated to obtain a more detailed understanding of the role of VPEs in plants.

Expression of a Small Ubiquitin-Like Modifier Protease Increases Drought Tolerance in Wheat (Triticum aestivum L.).

Post-translation modification of proteins plays a critical role in cellular signaling processes. In recent years, the SUMO (Small Ubiquitin-Like Modifier) class of molecules has emerged as an influential mechanism for target protein management. SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responses. In the present study, the expression of an Arabidopsis thaliana cysteine protease (OVERLY TOLERANT TO SALT-1, OTS1) in wheat (Triticum aestivum L.) has led to improved plant growth under water stress conditions. Transformed wheat (pUBI-OTS1) displayed enhanced growth and delayed senescence under water deficit when compared with untransformed Gamtoos-R genotype or plants carrying an empty vector. Transformed pUBI-OTS1 plants also maintained a high relative moisture content (RMC), had a higher photosynthesis rate, and also had a higher total chlorophyll content when compared to untransformed plants or plants carrying an empty vector. SUMOylation of total protein also increased in untransformed plants but not in the AtOTS1 transformed plants. Our results suggest that SUMO-proteases may influence an array of mechanisms in wheat to the advantage of the crop to be more tolerant to water stress caused by drought. This is the first report to elucidate SUMOylation effects in the hexaploid crop wheat (T. aestivum L.).

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.

Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association.

Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease-mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions.

Enhancing faba bean (Vicia faba L.) genome resources.

Grain legume improvement is currently impeded by a lack of genomic resources. The paucity of genome information for faba bean can be attributed to the intrinsic difficulties of assembling/annotating its giant (~13 Gb) genome. In order to address this challenge, RNA-sequencing analysis was performed on faba bean (cv. Wizard) leaves. Read alignment to the faba bean reference transcriptome identified 16 300 high quality unigenes. In addition, Illumina paired-end sequencing was used to establish a baseline for genomic information assembly. Genomic reads were assembled de novo into contigs with a size range of 50-5000 bp. Over 85% of sequences did not align to known genes, of which ~10% could be aligned to known repetitive genetic elements. Over 26 000 of the reference transcriptome unigenes could be aligned to DNA-sequencing (DNA-seq) reads with high confidence. Moreover, this comparison identified 56 668 potential splice points in all identified unigenes. Sequence length data were extended at 461 putative loci through alignment of DNA-seq contigs to full-length, publicly available linkage marker sequences. Reads also yielded coverages of 3466× and 650× for the chloroplast and mitochondrial genomes, respectively. Inter- and intraspecies organelle genome comparisons established core legume organelle gene sets, and revealed polymorphic regions of faba bean organelle genomes.

A novel inversion in the chloroplast genome of marama (Tylosema esculentum).

Tylosema esculentum (marama bean) is being developed as a possible crop for resource-poor farmers in arid regions of Southern Africa. As part of the molecular characterization of this species, the chloroplast genome has been assembled from next-generation sequencing using both Illumina and Pac-Bio data. The genome is of typical organization with a large single-copy region and a small single-copy region separated by a pair of inverted repeats and covers 161537 bp. It contains a unique inversion not present in any other legumes, even in the closest relatives for which the complete chloroplast genome is available, and two complete copies of the ycf1 gene. These data extend the range of variability of legume chloroplast genomes. The sequencing of multiple individuals has identified two different chloroplast genomes which were geographically separated. The current sampling is limited so that the extent of the intraspecific variation is still to be determined, leaving open the question of legume chloroplast genomes adapted to particular arid environments.

Unlocking the potential of orphan legumes.

Orphan, or underutilized, legumes are domesticated legumes with useful properties, but with less importance than major world crops due to use and supply constraints. However, they play a significant role in many developing countries, providing food security and nutrition to consumers, as well as income to resource-poor farmers. They have been largely neglected by both researchers and industry due to their limited economic importance in the global market. Orphan legumes are better adapted than the major legume crops to extreme soil and climatic conditions, with high tolerance to abiotic environmental stresses such as drought. As a stress response they can also produce compounds with pharmaceutical value. Orphan legumes are therefore a likely source of important traits for introduction into major crops to aid in combating the stresses associated with global climate change. Modern large-scale genomics techniques are now being applied to many of these previously understudied crops, with the first successes reported in the genomics area. However, greater investment of resources and manpower are necessary if the potential of orphan legumes is to be unlocked and applied in the future.

Agroinfiltration contributes to VP1 recombinant protein degradation.

There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

Review: The future of cystatin engineering.

Plant cystatins are naturally occurring protease inhibitors that prevent proteolysis by papain-like cysteine proteases. Their protective action against environmental stresses has been relatively well characterised. Still, there is a need to greatly improve both potency and specificity based on the current rather poor performance of cystatins in biotechnological applications. Research in creating more potent and specific cystatins, including amino acid substitutions in either conserved cystatin motifs and/or at variable amino acid sites, is reviewed. Existing gaps for better understanding of cystatin-protease interactions are further explored. Current knowledge on multi-cystatins or hybrid protease inhibitors involving cystatins as an additional option for cystatin engineering is further outlined along with the nuances of how cystatins with rather unusual amino acid sequences might actually help in cystatin engineering. Finally, future opportunities for application of cystatins are highlighted which include applications in genetically modified transgenic plants for environmental stress protection and also as nutraceuticals, as part of more nutritious food. Further opportunities might also include the possible management of diseases and disorders, often associated with lifestyle changes, and the most immediate and promising application which is inclusion into plant-based recombinant protein production platforms.

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.

Potential use of phytocystatins in crop improvement, with a particular focus on legumes.

Phytocystatins are a well-characterized class of naturally occurring protease inhibitors that function by preventing the catalysis of papain-like cysteine proteases. The action of cystatins in biotic stress resistance has been studied intensively, but relatively little is known about their functions in plant growth and defence responses to abiotic stresses, such as drought. Extreme weather events, such as drought and flooding, will have negative impacts on the yields of crop plants, particularly grain legumes. The concepts that changes in cellular protein content and composition are required for acclimation to different abiotic stresses, and that these adjustments are achieved through regulation of proteolysis, are widely accepted. However, the nature and regulation of the protein turnover machinery that underpins essential stress-induced cellular restructuring remain poorly characterized. Cysteine proteases are intrinsic to the genetic programmes that underpin plant development and senescence, but their functions in stress-induced senescence are not well defined. Transgenic plants including soybean that have been engineered to constitutively express phytocystatins show enhanced tolerance to a range of different abiotic stresses including drought, suggesting that manipulation of cysteine protease activities by altered phytocystatin expression in crop plants might be used to improve resilience and quality in the face of climate change.

Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.