Pod Tissue Research Articles

Microscopical Analysis of Autofluorescence as a Complementary and Useful Method to Assess Differences in Anatomy and Structural Distribution Underlying Evolutive Variation in Loss of Seed Dispersal in Common Bean.

The common bean has received attention as a model plant for legume studies, but little information is available about the morphology of its pods and the relation of this morphology to the loss of seed dispersal and/or the pod string, which are key agronomic traits of legume domestication. Dehiscence is related to the pod morphology and anatomy of pod tissues because of the weakening of the dorsal and ventral dehiscence zones and the tensions of the pod walls. These tensions are produced by the differential mechanical properties of lignified and non-lignified tissues and changes in turgor associated with fruit maturation. In this research, we histologically studied the dehiscence zone of the ventral and dorsal sutures of the pod in two contrasting genotypes for the dehiscence and string, by comparing different histochemical methods with autofluorescence. We found that the secondary cell wall modifications of the ventral suture of the pod were clearly different between the dehiscence-susceptible and stringy PHA1037 and the dehiscence-resistant and stringless PHA0595 genotypes. The susceptible genotype had cells of bundle caps arranged in a more easily breakable bowtie knot shape. The resistant genotype had a larger vascular bundle area and larger fibre cap cells (FCCs), and due to their thickness, the external valve margin cells were significantly stronger than those from PHA1037. Our findings suggest that the FCC area, and the cell arrangement in the bundle cap, might be partial structures involved in the pod dehiscence of the common bean. The autofluorescence pattern at the ventral suture allowed us to quickly identify the dehiscent phenotype and gain a better understanding of cell wall tissue modifications that took place along the bean's evolution, which had an impact on crop improvement. We report a simple autofluorescence protocol to reliably identify secondary cell wall organization and its relationship to the dehiscence and string in the common bean.

Fine mapping and characterisation of a PV-PUR mediating anthocyanin synthesis in snap bean (Phaseolus vulgaris L.).

The online version contains supplementary material available at 10.1007/s11032-023-01362-8.

Responses of Phosphorus Applications in Two Cultivars of Thailand’s Dwarf Yard-Long Beans

Phosphorus (P) fertilizer applications have been determined to greatly improve the growth and yield of dwarf yard-long beans. If the plants receive an insufficient amount of P, growth and yield may be reduced. We conducted a pot experiment to investigate the responses of two cultivars of dwarf yard-long beans (KKU25 and CLGC30), and the effects of the application of P fertilizer (0 and 150 kg/ha) on the growth, yield, nutrient values, P uptake and partitioning after harvesting in two soil series [Tha rua (Tr) and Ayutthaya (Ay) soil]. Our results demonstrated that the application of P fertilizer in soil with low available P (Tr soil) produced a significant difference in the yield and yield component of the plants. Pod tissue was an important source of P in the plants at maturity. The results also showed that over 40% of P was accumulated in the pod tissue. The CLGC30 variety showed higher total P uptake in plant tissue than that of the KKU25. It was also found that the application of P fertilizer gave a higher total P uptake than the control. Above-ground biomass (AB) and above-ground P content (APC) were significantly affected by cultivar with the application of P fertilizer. CLGC30 with applied P fertilizer produced higher AB and APC values than that of KKU25 without the application of P fertilizer. Consequently, the application of P fertilizer for dwarf yard-long bean production is recommended for the application of soil with low available P.

Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues.

Vanilla is a globally important spice crop used in a variety of food, cosmetic, and pharmaceutical products. V. planifolia is the primary commercial species with V. x tahitensis also permissible for food use. Other aromatic species, including V. pompona, are used for food throughout Central and South America. Supply chain complexity hinders the vanilla bean industry and can lead to false claims of genetic and geographical origins to obtain higher prices. Beans of some species can be differentiated by experienced buyers, but hybrids and morphological differences caused by environmental variability or disease would best be resolved by diagnostic tests. METHODS ANDRESULTS: Kompetitive Allele Specific Polymerase Chain Reaction is a widely used molecular marker that can genotype single nucleotide polymorphisms efficiently and inexpensively. Assays were designed to differentiate V. planifolia, V. x tahitensis, and V. pompona using publicly available vanilla genomics data. Ten KASP assays on chromosomes 1 through 7, the ITS region, and plastid-encoded rbcL gene successfully differentiated V. planifolia, V. odorata, and V. x tahitensis. Additional KASP assays on chromosomes 1 through 4, the ITS region, and rbcL gene successfully differentiated V. planifolia and V. pompona. Further, a method for extracting KASP-quality DNA from cured vanilla bean seeds was developed and successfully differentiated V. planifolia, V. odorata, V. x tahitensis, V. pompona, and their hybrids. The methods and results from this study can be used to identify interspecific hybrids, ensure the authenticity of cured vanilla beans, and reduce abuse within the vanilla supply chain.

Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

Feeding Behavior of Riptortus pedestris (Fabricius) on Soybean: Electrical Penetration Graph Analysis and Histological Investigations.

Simple SummaryRiptortus pedestris (Fabricius) (Hemiptera: Alydidae) is a major soybean pest with the peak population occurrence during the seed maturity stage from pod filling to harvest. Soybean pods and/or seeds are required for R. pedestris development. However, the feeding strategies employed by this stink bug to feed on soybean are still not clear. In the present study, we recorded the feeding behaviors of R. pedestris on soybean using electropenetrography (EPG). The biological meaning of each waveform was confirmed by histological examination of plant tissues containing stylets or salivary sheath. In total, five phases of waveforms were identified: non-probing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4). Xylem ingestion (Rp2) was observed during R. pedestris feeding on soybean leaflets, stems, and pods, demonstrating that the stink bug was ingesting xylem sap from vascular tissue. Cell rupture (salivation/ingestion, Rp3) was only recorded during R. pedestris feeding on cotyledon and pods. Histological images showed that stylet tips and the salivary sheath were positioned in the tissues of cotyledon and pods. Taken together, our results demonstrate that R. pedestris uses a cell-rupture strategy to acquire nutrients from soybean pods and/or seeds while utilizing salivary sheath tactics to obtain water from xylem sap. These findings provide insightful information to understand the interactions between R. pedestris and the soybean plant. Riptortus pedestris (Fabricius) is a major agricultural pest feeding on soybean pods and seeds. The large populations occur during seed maturity stages from pod filling to harvest. Its infestation results in shriveled and dimpled seeds while vegetative structures (leaflet and stem) remain green, known as “Stay Green” syndrome. Additional evidence also demonstrates that soybean pods and seeds are required for Riptortus pedestris development. However, the feeding behavior strategies employed by this stink bug to feed on soybean plants are still not clear. In the present study, the feeding behaviors of R. pedestris on soybean plants were recorded by electropenetrography (EPG), and a waveform library was created for this species. A total of five phases of waveforms—nonprobing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4)—were identified. Non-probing waveforms Z and NP and pathway (Rp1) were found in all tested plant structures (leaflet, stem, cotyledon, and pods). Waveform Rp2 (xylem sap ingestion, xylem ingestion) was primarily recorded during R. pedestris feeding on leaflets and stems, while Rp3 (salivation/ingestion) was only observed during feeding on cotyledon and pods. Histological examinations confirmed that correlation between Rp2 and stylet tip positioning in the xylem vessel in leaflets and stems. Stylet tips end in the tissues of cotyledon and pods when Rp3 is recorded. Taken together, our results demonstrate that R. pedestris ingests xylem sap from vegetative tissues of soybean (leaflet and stem) via a salivary sheath strategy to obtain water. It mainly acquires nutrients from soybean pods and/or seeds using cell-rupture tactics. This study provided insightful information to understand the field occurrence patterns of “Stay Green” syndrome, which may have important implications for pest control.

Investigating Nutrient Supply Effects on Plant Growth and Seed Nutrient Content in Common Bean.

Low soil fertility commonly limits growth and yield production of common bean (Phaseolus vulgaris L.) in tropical regions. Impacts of nutrient limitations on production volume are well studied and are a major factor in reducing crop yields. This study characterised the impact of reduced nutrient supply on carbon assimilation and nutrient content of leaf, phloem sap and reproductive tissues of common bean grown in a controlled environment in order to detect chemical markers for changes in nutritional content. Leaf gas exchange measurements were undertaken over plant development to characterise changes to carbon assimilation under reduced nutrient supply. Samples of leaf, phloem sap and pod tissue of common bean were analysed for carbon isotope discrimination, mineral nutrient content, and amino acid concentration. Despite declines in nutrient availability leading to decreased carbon assimilation and reductions in yield, amino acid concentration was maintained in the pod tissue. Common bean can maintain the nutritional content of individual pods under varying nutrient availabilities demonstrating the resilience of processes determining the viability of reproductive tissues.

Changes in Sugar Concentrations of Seed and Pod Tissue During Development in Snap and Dry Beans (Phaseolus vulgaris L.)

Sugars, including glucose, fructose, and sucrose, contribute significantly to the flavor and consumer acceptance of snap beans (Phaseolus vulgaris L.). Sugar accumulation and changes in sugar profiles during snap bean development contribute to overall assessments of quality for breeding lines and cultivars. Developing fruit from a diverse group of four snap bean cultivars containing Andean germplasm and one Mesoamerican dry bean cultivar were sampled at 5-day intervals from 10 to 30 days after flowering over 2 years. Glucose, fructose, and sucrose in pod and seed tissue was quantified using high-performance liquid chromatography. Percent seed mass relative to pod mass increased with days after flowering, but the rate of increase was heterogeneous among cultivars. Significant differences in sugar accumulation patterns of mono- and disaccharides were observed with time of development and between pods and seeds. Glucose and fructose decreased rapidly in pods and seeds with time after flowering. In contrast, sucrose concentration increased in pod tissue but remained constant in seeds of the snap bean cultivars with time after flowering. The patterns of changes in pod and seed sugar concentrations with time after flowering were similar among all snap bean cultivars. In contrast to the snap beans, seed sucrose increased with time after flowering in the Mesoamerican dry bean cultivar Puebla 152. No year by day after flowering interactions were observed for sugar accumulation patterns or sugar concentrations. Younger snap beans had the highest sweetness index based on observed sugar concentrations, percent seed mass, and perception of relative sweetness by the human palate. Although mean sweetness varied between cultivars, the rate of decrease in sweetness with time was the same for all five cultivars. These findings indicate that variation for sweetness exists in snap beans and can be exploited by breeding to develop cultivars with a potentially more desirable, sweet flavor.

Morphological, molecular and pathogenic characterization of Phytophthora palmivora isolates causing black pod rot of cacao in Colombia

Aim of study: To characterize isolates of Phytophthora sp. causing black pod rot (BPR) of cacao (Theobroma cacao L.).Area of study: Eight cocoa-growing regions in Colombia.Material and methods: Sixty isolates of Phytophthora sp. were obtained from tissues of cacao pods showing symptoms of BPR. Isolates were characterized using the morphology of sporangia and chlamydospores, molecular sequencing of regions of nuclear DNA (rDNA-ITS) and mitochondrial (COX) and virulence in different genotypes of cocoa pods.Main results: A high phenotypic variability between the isolates was determined, being the pedicel length and the length/width ratio (L/W) the most stable characters for species identification. Short pedicels with an average of 3.13 μm ± 0.28 and a length/width ratio of sporangia (L/W) with an average of 1.55 μm ± 0.11 were established as the most consistent morphological characteristics within palmivora species.Research highlights: Phytophthora pamivora was the only species associated to BPR, identified using morphology together with sequence analyses.

Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue.

Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.

Antifungal Efficacy of Crude Aqueous Weed Extracts Against Pathogen of Cocoa Black Pod Rot

Black pod rot is the most economically important disease of cocoa in Malaysia which is mainly caused by a highly polyphagous Phytophthora species, called Phytophthora palmivora. The fungus could attack all parts of the cocoa plant organs and caused various diseases at any growth stage from seedling until the mature stages, especially during raining season. The application of synthetic fungicides has been widely recommended to manage the disease but their repeated use had led to other problems such as environmental, human health and development of fungicide resistance issues. This study isolated and identified Phytopththora isolate from a cocoa pod sample based on micro-morphological characters. Besides, the present investigation was undertaken to screen for the antifungal potency of different weed extracts against the Phytophthora pathogen using poisoned food technique. The fungal isolate was successfully recovered from pod tissues of clone PBC123 on 20% tomato juice agar culture (20T). Only one out of ten weed extracts tested showed a significant in vitro inhibitory effect towards mycelial growth of Phytophthora isolate, which was aqueous crude leaf extract of Solanum torvum (42.68%). This study indicated that the potential of weed extracts in the management of Phytophthora diseases, and may offer more natural, effective and economical control methods.

Nutritional restriction triggers callose accumulation on the sieve plates of the funiculus of developing bean seeds

Seed development represents a very high investment of metabolic resources that plants are not always able to fulfill, and nutrient remobilization is widely used to support seed development under stressful conditions. Phaseolus vulgaris fruits removed from the plant at 20 days after anthesis (DAA) respond to nutrient restriction by recycling pod material. At the same time, the available resources are channeled to a few seeds in order to produce some viable seeds. By analyzing carbohydrate levels and the distribution of sucrose [U-14C] between the pod tissue and seed sections (funiculus, seed coat and embryo), in combination with the structural analysis of phloem sieve plates; we were able to identify that nutrient flow is reduced at the funiculus level. The accumulation of callose resulted in a large reduction in the diameter of the sieve pores that eventually will stop nutrient flow. It produces the death of most seeds, but also increases the probability of some seeds to successfully completing their development during severe nutrient restriction.

Low N Fertilizer Application and Intercropping Increases N Concentration in Pea (Pisum sativum L.) Grains.

Sustainable intensification of pulses needs reduced input of nitrogen (N) fertilizer with enhanced crop nutritional quality and yield. Therefore, increasing N harvest in grains (sink organs) by improving N remobilization is of key importance. Previous research has shown that a lower dose of N fertilizer effectively increases the rate of N remobilization, while intercropping improves the grain N concentration in pea (Pisum sativum L.). However, it is unknown whether intercropping can facilitate this N fertilizer effect to increase N remobilization, and thereby enhance the N harvest index (NHI). In this study, we determined N allocation among different organs of pea plants, N translocation from leaf and stem tissues to pods, N2 fixation, N utilization efficiency, and NHI of pea plants grown alone or intercropped with maize (Zea mays L.) with different N fertilization treatments in a field experiment in northwestern China from 2012 to 2014. A base application of 90 kg N ha−1 at sowing and top-dress application of 45 kg N ha−1 at flowering integrated with maize–pea intercropping increased N allocation to pod tissues, N translocation to grains, and NHI of pea plants. Compared with the application of 90 kg N ha−1 at sowing and 135 kg N ha−1 top-dressed at flowering, reducing the top-dress application of N fertilizer to 45 kg N ha−1 increased N allocation to intercropped pea plants by 8%. Similarly, N translocation to grains from leaf and stem tissues was increased by 37.9 and 43.2%, respectively, enhancing the NHI by 40.1%. A positive correlation between N2 fixation and NHI was observed, implying that N2 fixation improves N concentration in grain sinks. Thus, our data show that growing pulses in an intercropping system with reduced N fertilization are essential for maximizing N translocation, improving nutritional quality, and preventing the loss of N through leaching, thereby avoiding potential groundwater contamination.

Electropenetrography Monitoring of the Neotropical Brown-Stink Bug (Hemiptera: Pentatomidae) on Soybean Pods: An Electrical Penetration Graph-Histology Analysis.

The Neotropical brown-stink bug Euschistus heros (F.) (Hemiptera: Pentatomidae) is the most important pest damaging soybean in the Neotropics, the world largest production area. The alternating current–direct current (AC–DC) electropenetrography (EPG) technology was used to develop an EPG waveform library of adult females feeding on soybean (Glycine max (L.) Merr.) pods at different input resistor (Ri) levels. Thirteen waveform types/subtypes were identified and described. They were divided into non-probing (Z, Np, Dw1, and Dw2), and probing waveforms (Eh1, Eh2, Eh3, Eh4, and Eh5). Probing waveforms were grouped into three phases: 1) pathway (Eh1a, Eh1b, Eh1c, and Eh1w), 2) ingestion (Eh2, Eh3a, Eh3b, and Eh4), and 3) interruption (Eh5). Correlations between waveforms Eh1b, Eh1c, Eh2, Eh3, and Eh4 and stylets tip position and/or salivary sheath in the pod tissue were determined via histological studies. Non-probing waveforms, Z and Np were visually associated with the bug resting and walking on pod surface, respectively. Waveform Dw1 was correlated with egestion, and the ingestion of fluids (droplets) was proposed for Dw2. Eh1a and Eh1b corresponded to initial and deep stylet penetration through pod tissue, and secretion of a salivary sheath. In Eh1c, stylets penetrated the rigid cell layer of sclerenchyma, and during Eh1w they were withdrawn. Eh2 represents sustained xylem sap ingestion. Eh3a corresponded to lacerate and macerate cell rupture feeding behavior in seed endosperm, whereas Eh3b corresponded to ingestion of cellular contents. Eh4 represented short ingestion from an unknown site, and Eh5 represented short interruptions during xylem sap ingestion.

GmWRKY49, a Salt-Responsive Nuclear Protein, Improved Root Length and Governed Better Salinity Tolerance in Transgenic Arabidopsis.

Plant WRKY transcription factors (TFs) are active guardians against pathogens’ insurgency, key components in developmental processes, contributors in signal transduction pathways, and regulators of diverse biotic and abiotic stress responses. In this research, we isolated, cloned, and functionally characterized a new WRKY TF GmWRKY49 from soybean. GmWRKY49 is a nuclear protein which contains two highly conserved WRKY domains and a C2H2-type zinc-finger structure. The normalized expression (log2 ratio) of GmWRKY49 was 2.75- and 1.90-fold in salt-tolerant and salt-susceptible soybean genotypes, respectively. The transcripts of GmWRKY49 could be detected in roots, stems, leaves, flowers, and almost no expression in pod tissues. The salinity-tolerance response of this gene was studied through overexpression in soybean composite seedlings and transgenic Arabidopsis. The effect of GmWRKY49 overexpression on root length of transgenic Arabidopsis was also investigated. Under salt stress, several parameters including germination rate, survival rate, root length, rosette diameter, relative electrolyte leakage, and proline content were significantly higher in composite seedlings and transgenic Arabidopsis than those in wild-type. Moreover, GmWRKY49 enhanced salinity tolerance in soybean mosaic seedlings and transgenic Arabidopsis. These results suggest that GmWRKY49 is a positive regulator of salinity tolerance in soybean and has high potential utilization for crop improvement.

Accumulation of anthocyanin in callus cultures of red-pod okra [Abelmoschus esculentus (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m− 2 s− 1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47 ± 0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m− 2 s− 1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.

Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers

BackgroundThe medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson’s drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions.ResultsOne-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23 M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways.ConclusionsThe M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

A comparison of tissue preparation methods for protein extraction of cocoa (Theobroma cacao L.) pod

Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B) of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D). Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Method s B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.

Novel Genomic and Evolutionary Insight of WRKY Transcription Factors in Plant Lineage.

The evolutionarily conserved WRKY transcription factor (TF) regulates different aspects of gene expression in plants, and modulates growth, development, as well as biotic and abiotic stress responses. Therefore, understanding the details regarding WRKY TFs is very important. In this study, large-scale genomic analyses of the WRKY TF gene family from 43 plant species were conducted. The results of our study revealed that WRKY TFs could be grouped and specifically classified as those belonging to the monocot or dicot plant lineage. In this study, we identified several novel WRKY TFs. To our knowledge, this is the first report on a revised grouping system of the WRKY TF gene family in plants. The different forms of novel chimeric forms of WRKY TFs in the plant genome might play a crucial role in their evolution. Tissue-specific gene expression analyses in Glycine max and Phaseolus vulgaris showed that WRKY11-1, WRKY11-2 and WRKY11-3 were ubiquitously expressed in all tissue types, and WRKY15-2 was highly expressed in the stem, root, nodule and pod tissues in G. max and P. vulgaris.

Stochastic alternative splicing is prevalent in mungbean (Vigna radiata)

SummaryAlternative splicing (AS) can produce multiple mature mRNAs from the same primary transcript, thereby generating diverse proteins and phenotypes from the same gene. To assess the prevalence of AS in mungbean (Vigna radiata), we analysed whole‐genome RNA sequencing data from root, leaf, flower and pod tissues and found that at least 37.9% of mungbean genes are subjected to AS. The number of AS transcripts exhibited a strong correlation with exon number and thus resembled a uniform probabilistic event rather than a specific regulatory function. The proportion of frameshift splicing was close to the expected frequency of random splicing. However, alternative donor and acceptor AS events tended to occur at multiples of three nucleotides (i.e. the codon length) from the main splice site. Genes with high exon number and expression level, which should have the most AS if splicing is purely stochastic, exhibited less AS, implying the existence of negative selection against excessive random AS. Functional AS is probably rare: a large proportion of AS isoforms exist at very low copy per cell on average or are expressed at much lower levels than default transcripts. Conserved AS was only detected in 629 genes (2.8% of all genes in the genome) when compared to Vigna angularis, and in 16 genes in more distant species like soya bean. These observations highlight the challenges of finding and cataloguing candidates for experimentally proven AS isoforms in a crop genome.