Samuel Bocobza

Biosynthesis of Antinutritional Alkaloids in Solanaceous Crops Is Mediated by Clustered Genes

From Nasty to Tasty Some of our favorite food crops derive from wild relatives that were distasteful or even toxic. Domestication over many years selected for variants with reduced levels of antinutritional compounds. The wild relatives remain valuable, however, for other traits such as resistance to pathogens, but their use in crop development is complicated by the continued presence of unpalatable compounds. Itkin et al. (p. 175, published online 20 June) elucidate the metabolic pathways and genes directing synthesis of some of these antinutritionals in potato and tomato. Some of the chemicals that domestication has reduced in potato and tomato are derived from clusters of biosynthetic genes. Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants—as, for example, potato—are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

The Arabidopsis DSO/ABCG11 Transporter Affects Cutin Metabolism in Reproductive Organs and Suberin in Roots

Apart from its significance in the protection against stress conditions, the cuticular cover is essential for proper development of the diverse surface structures formed on aerial plant organs. This layer mainly consists of a cutin matrix, embedded and overlaid with cuticular waxes. Following their biosynthesis in epidermal cells, cutin and waxes were suggested to be exported across the plasma membrane by ABCG-type transporters such as DSO/ABCG11 to the cell wall and further to extracellular matrix. Here, additional aspects of DSO/ABCG11 function were investigated, predominantly in reproductive organs, which were not revealed in the previous reports. This was facilitated by the generation of a transgenic DSO/ABCG11 silenced line (dso-4) that displayed relatively subtle morphological and chemical phenotypes. These included altered petal and silique morphology, fusion of seeds, and changes in levels of cutin monomers in flowers and siliques. The dso-4 phenotypes corresponded to the strong DSO/ABCG11 gene expression in the embryo epidermis as well as in the endosperm tissues of the developing seeds. Moreover, the DSO/ABCG11 protein displayed polar localization in the embryo protoderm. Transcriptome analysis of the dso-4 mutant leaves and stems showed that reduced DSO/ABCG11 activity suppressed the expression of a large number of cuticle-associated genes, implying that export of cuticular lipids from the plasma membrane is a rate-limiting step in cuticle metabolism. Surprisingly, root suberin composition of dso-4 was altered, as well as root expression of two suberin biosynthetic genes. Taken together, this study provides new insights into cutin and suberin metabolism and their role in reproductive organs and roots development.

Switching the light on plant riboswitches.

Riboswitches are natural RNA sensors that affect post-transcriptional processes via their capacity to bind small molecules. To date, these mRNA structures have been shown to regulate the biosynthesis of essential metabolites, including vitamins and amino acids. Although bacterial riboswitches are widespread and characterized, only a single eukaryotic, thiamin-pyrophosphate-binding riboswitch has recently been discovered to direct gene expression by regulating mRNA splicing in fungi, green algae and land plants. It is unclear how widespread riboswitches are and what additional roles they have in eukaryotes. When engineered in plants, riboswitches can function autonomously to modulate gene expression. These discoveries not only trigger novel findings regarding RNA switches in plants, but also spur the exploitation of riboswitches for monitoring metabolite concentrations in planta.

Orchestration of Thiamin Biosynthesis and Central Metabolism by Combined Action of the Thiamin Pyrophosphate Riboswitch and the Circadian Clock in Arabidopsis

This study reports on the physiological role of the TPP riboswitch noncoding RNA element in balancing thiamin levels, plant metabolism, and overall organismal fitness. The model suggests that in Arabidopsis, the THIC promoter and the riboswitch simultaneously tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism. Riboswitches are natural RNA elements that posttranscriptionally regulate gene expression by binding small molecules and thereby autonomously control intracellular levels of these metabolites. Although riboswitch-based mechanisms have been examined extensively, the integration of their activity with global physiology and metabolism has been largely overlooked. Here, we explored the regulation of thiamin biosynthesis and the consequences of thiamin pyrophosphate riboswitch deficiency on metabolism in Arabidopsis thaliana. Our results show that thiamin biosynthesis is largely regulated by the circadian clock via the activity of the THIAMIN C SYNTHASE (THIC) promoter, while the riboswitch located at the 3′ untranslated region of this gene controls overall thiamin biosynthesis. Surprisingly, the results also indicate that the rate of thiamin biosynthesis directs the activity of thiamin-requiring enzymes and consecutively determines the rate of carbohydrate oxidation via the tricarboxylic acid cycle and pentose-phosphate pathway. Our model suggests that in Arabidopsis, the THIC promoter and the thiamin-pyrophosphate riboswitch act simultaneously to tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism.

The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism.

Steroidal alkaloids (SAs) and their glycosylated forms (SGAs) are toxic compounds largely produced by members of the Solanaceae and Liliaceae plant families. This class of specialized metabolites serves as a chemical barrier against a broad range of pest and pathogens. In humans and animals, SAs are considered anti-nutritional factors because they affect the digestion and absorption of nutrients from food and might even cause poisoning. In spite of the first report on SAs nearly 200 years ago, much of the molecular basis of their biosynthesis and regulation remains unknown. Aspects concerning chemical structures and biological activities of SAs have been reviewed extensively elsewhere; therefore, in this review the latest insights to the elucidation of the SAs biosynthetic pathway are highlighted. Recently, co-expression analysis combined with metabolic profiling revealed metabolic gene clusters in tomato and potato that contain core genes required for production of the prominent SGAs in these two species. Elaborating the knowledge regarding the SAs biosynthetic pathway, the subcellular transport of these molecules, as well as the identification of regulatory and signaling factors associated with SA metabolism will likely advance understanding of chemical defense mechanisms in Solanaceae and Liliaceae plants. It will also provide the means to develop, through classical breeding or genetic engineering, crops with modified levels of anti-nutritional SAs.

Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae

Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt.

Discovery of new modules in metabolic biology using ChemoMetabolomics.

The use of small molecules to transiently modulate protein function can circumvent the limitations of classical genetic approaches caused by gene redundancy and lethality. Although chemical genomics and genetics screens facilitate the characterization of new biological components, they have infrequently led to the identification of target proteins involved in metabolism. The current state of metabolomics technologies permits the detection of thousands of molecules, allowing the exploration of yet uncharacterized metabolic pathways. The combination of these two approaches, termed here “ChemoMetabolomics”, is a promising application of both technologies. This novel approach will facilitate the detection of metabolic modulators, the dissection of the crosstalk in the metabolic network, and the development of hypotheses on how changes in metabolism affect developmental or cellular responses. Furthermore, it will facilitate the elucidation of the linkage between metabolic and developmental programs and assist the gain of an elaborated view of biological processes at the system level.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum

Significance Plants synthesize a vast repertoire of steroidal specialized metabolites. These include the well-known class of antinutritional steroidal glycoalkaloids (SGAs), which act as defensive chemicals in the Solanaceae, and the pharmacologically important and widespread steroidal saponins. Here, we uncover an elusive enzymatic step that acts on unsaturated steroidal metabolites. We find that GLYCOALKALOID METABOLISM25 (GAME25) acts at a key branch point in the biosynthesis pathways of steroidal specialized metabolites. The activity of GAME25 not only affects the enormous diversity of SGAs and steroidal saponins, which are produced in hundreds of plant species, but also modulates the molecules’ toxic effects. This work helps explain the extensive structural diversity in specialized metabolism through a relatively simple chemical modification in a single metabolite backbone. Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.