Cookability of 24 pea accessions-determining factors and potential predictors of cooking quality.

BACKGROUND: Cooking time and cooking evenness are two critical quantities when determining the cooking quality (termed cookability) of pulses. Deciphering which factors contribute to pulse cookability is important for breeding new cultivars, and the identification of potential cookability predictors can facilitate breeding efforts. Seeds from 24 morphologically diverse pea accessions were tested to identify contributing factors and potential predictors of the observed cookability using a Mattson cooker. Size- and weight-based measures were recorded, and seed-coat hardness was obtained with a penetrometer. Content of protein, starch (amylose and amylopectin), and phytate was also determined. RESULTS: Distinct differences were found between wrinkled and non-wrinkled seeds in terms of water-absorption capacity, seed-coat hardness, and plunger-perforation speed. Potential predictive indicators of cooking time and cooking evenness were seed-coat hardness (r = 0.49 and r = 0.38), relative area gained (r = -0.59 and r = -0.8), and percentage of swelled seeds after soaking (r = -0.49 and r = -0.58), but only for non-wrinkled seeds. Surprisingly, the coefficients of variation for the profile area of both dry and swelled seeds appeared to be potential cookability predictors of all pea types (correlation coefficients around r = 0.5 and supported by principal component analysis). However, no strong correlation was observed between cookability and protein, starch, or phytate levels. CONCLUSION: Using three types of instruments together with chemical components enabled the identification of novel cookability predictors for both cooking time and cooking evenness in pea. This study unveils the diverse quantitative aspects influencing cookability in pea. Considering both cooking time and cooking evenness, as well as seed-coat hardness, underscores the multifaceted nature of pulse cookability and offers important insights for future breeding strategies to enhance pea cultivars. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin.

BACKGROUND: Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV). RESULTS: We identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)-a gene likely to be involved in toxic alkaloid biosynthesis-we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a 'PDS co-silencing' approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL. CONCLUSIONS: We provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

In planta and in silico characterization of five sesquiterpene synthases from Vitis vinifera (cv. Shiraz) berries.

MAIN CONCLUSION: Five Vitis vinifera sesquiterpene synthases were characterized, two was previously uncharacterized, one being a caryophyllene/cubebene synthase and the other a cadinene synthase. Residue differences with other Vitis sesquiterpene synthases are described. The biochemical composition of grape berries at harvest can have a profound effect on the varietal character of the wine produced. Sesquiterpenes are an important class of volatile compounds produced in grapes that contribute to the flavor and aroma of wine, making the elucidation of their biosynthetic origin an important field of research. Five cDNAs corresponding to sesquiterpene synthase genes (TPSs) were isolated from Shiraz berries and expressed in planta in Nicotiana benthamiana followed by chemical characterization by GC-MS. Three of the TPS cDNAs were isolated from immature berries and two were isolated from ripe Shiraz berries. Two of the investigated enzymes, TPS26 and TPS27, have been previously investigated by expression in E. coli, and the in planta products generally correspond to these previous studies. The enzyme TPS07 differed by eight amino acids (none of which are in the active site) from germacrene B and D synthase isolated from Gewürztraminer grapes and characterized in vitro. Here in planta characterization of VvShirazTPS07 yielded ylangene, germacrene D and several minor products. Two of the enzymes isolated from immature berries were previously uncharacterized enzymes. VvShirazTPS-Y1 produced cadinene as a major product and at least 17 minor sesquiterpenoid skeletons. The second, VvShirazTPS-Y2, was characterized as a caryophyllene/cubebene synthase, a combination of products not previously reported from a single enzyme. Using in silico methods, we identified residues that could play key roles regarding differences in product formation of these enzymes. The first ring closure that is either a 1,10- or 1,11-ring closure is likely controlled by three neighboring amino acids in helices G1, H2, and J. As for many other investigated TPS enzymes, we also observe that only a few residues can account for radical changes in product formation.

Evaluation of synthetic promoters in Physcomitrella patens.

Securing a molecular toolbox including diverse promoters is essential for genome engineering. However, native promoters have limitations such as the available number or the length of the promoter. In this work, three short synthetic promoters were characterized by using the yellow fluorescent protein Venus. All of the tested promoters were active and showed higher mRNA expression than housekeeping gene PpAct7, and similar protein expression level to the AtUBQ10 promoter. This study shows that few cis-regulatory elements are enough to establish a strong promoter for continuous expression of genes in plants. Along with this, the study enhance the number of available promotors to be used in P. patens. It also demonstrates the potential to construct multiple non-native promoters on demand, which would aid to resolve one bottleneck in multiple pathway expression in P. patens and other plants.

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

BACKGROUND: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects. RESULTS: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. CONCLUSION: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

Transcriptome analysis of Thapsia laciniata Rouy provides insights into terpenoid biosynthesis and diversity in Apiaceae.

Thapsia laciniata Rouy (Apiaceae) produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp). Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae.