Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Wheat gibberellin oxidase genes and their functions in regulating tillering.

Multiple genetic factors control tillering, a key agronomy trait for wheat (Triticum aestivum L.) yield. Previously, we reported a dwarf-monoculm mutant (dmc) derived from wheat cultivar Guomai 301, and found that the contents of gibberellic acid 3 (GA3) in the tiller primordia of dmc were significantly higher. Transcriptome analysis indicated that some wheat gibberellin oxidase (TaGAox) genes TaGA20ox-A2, TaGA20ox-B2, TaGA3ox-A2, TaGA20ox-A4, TaGA2ox-A10 and TaGA2ox-B10 were differentially expressed in dmc. Therefore, this study systematically analyzed the roles of gibberellin oxidase genes during wheat tillering. A total of 63 TaGAox genes were identified by whole genome analysis. The TaGAoxs were clustered to four subfamilies, GA20oxs, GA2oxs, GA3oxs and GA7oxs, including seven subgroups based on their protein structures. The promoter regions of TaGAox genes contain a large number of cis-acting elements closely related to hormone, plant growth and development, light, and abiotic stress responses. Segmental duplication events played a major role in TaGAoxs expansion. Compared to Arabidopsis, the gene collinearity degrees of the GAoxs were significantly higher among wheat, rice and maize. TaGAox genes showed tissue-specific expression patterns. The expressions of TaGAox genes (TaGA20ox-B2, TaGA7ox-A1, TaGA2ox10 and TaGA3ox-A2) were significantly affected by exogenous GA3 applications, which also significantly promoted tillering of Guomai 301, but didn't promote dmc. TaGA7ox-A1 overexpression transgenic wheat lines were obtained by Agrobacterium mediated transformation. Genomic PCR and first-generation sequencing demonstrated that the gene was integrated into the wheat genome. Association analysis of TaGA7ox-A1 expression level and tiller number per plant demonstrated that the tillering capacities of some TaGA7ox-A1 transgenic lines were increased. These data demonstrated that some TaGAoxs as well as GA signaling were involved in regulating wheat tillering, but the GA signaling pathway was disturbed in dmc. This study provided valuable clues for functional characterization of GAox genes in wheat.

Effect of postharvest nicotinamide treatment on NAD+ metabolism and redox status in strawberry fruit during storage.

The increased activity of PARP enzymes is associated with a deficiency of NAD+, as well as with a loss of NADPH and ATP, and consequent deterioration of the redox state in fruits. In this study, we checked whether treatment with nicotinamide (NAM) would affect PARP-1 expression and NAD+ metabolism in strawberry fruit during storage. For this purpose, strawberry fruits were treated with 10 mM NAM and co-treated with NAM and UV-C, and then stored for 5 days at 4 °C. Research showed that nicotinamide contributes to reducing oxidative stress level by reducing PARP-1 mRNA gene expression and the protein level resulting in higher NAD+ availability, as well as improving energy metabolism and NADPH levels in fruits, regardless of whether they are exposed to UV-C. The above effects cause fruits treated with nicotinamide to be characterised by higher anti-radical activity, and a lower level of reactive oxygen species in the tissue.

Disclosing the molecular basis of salinity priming in olive trees using proteogenomic model discovery.

Plant responses to salinity are becoming increasingly understood, however, salt priming mechanisms remain unclear, especially in perennial fruit trees. Herein, we showed that low-salt pre-exposure primes olive (Olea europaea) plants against high salinity stress. We then performed a proteogenomic study to characterize priming responses in olive roots and leaves. Integration of transcriptomic and proteomic data along with metabolic data revealed robust salinity changes that exhibit distinct or overlapping patterns in olive tissues, among which we focused on sugar regulation. Using the multi-crossed -omics data set, we showed that major differences between primed and nonprimed tissues are mainly associated with hormone signaling and defense-related interactions. We identified multiple genes and proteins, including known and putative regulators, that reported significant proteomic and transcriptomic changes between primed and nonprimed plants. Evidence also supported the notion that protein post-translational modifications, notably phosphorylations, carbonylations and S-nitrosylations, promote salt priming. The proteome and transcriptome abundance atlas uncovered alterations between mRNA and protein quantities within tissues and salinity conditions. Proteogenomic-driven causal model discovery also unveiled key interaction networks involved in salt priming. Data generated in this study are important resources for understanding salt priming in olive tree and facilitating proteogenomic research in plant physiology.

Calcium affects uranium responses in Arabidopsis thaliana: From distribution to toxicity.

Uranium, a heavy metal and primordial radionuclide, is present in surface waters and soils both naturally and due to industrial activities. Uranium is known to be toxic to plants and its uptake and toxicity can be influenced by multiple factors such as pH and the presence of different ions. However, the precise role of the different ions in uranium uptake is not yet known. Here we investigated whether calcium influences uranium uptake and toxicity in the terrestrial plant Arabidopsis thaliana. To this end, A. thaliana plants were exposed to different calcium and uranium concentrations and furthermore, calcium channels were blocked using the calcium channel blocker lanthanum chloride (LaCl3). Fresh weight, relative growth rate, concentration of nutrients and uranium and gene expression of oxidative stress-related genes and calcium transporters were determined in roots and shoots. Calcium affected plant growth and oxidative stress in both control (no uranium) and uranium-exposed plants. In shoots, this was influenced by the total calcium concentration, but not by the different tested uranium concentrations. Uranium in turn did influence calcium uptake and distribution. Uranium-exposed plants grown in a medium with a higher calcium concentration showed an increase in gene expression of NADPH oxidases RBOHC and RBOHE and calcium transporter CAX7 after uranium exposure. In roots, these calcium-dependent responses in gene expression were not observed. This indicates that calcium indeed affects uranium toxicity, but only in shoots. In addition, a clear influence of uranium and LaCl3 (separately and combined) on the expression of calcium transporters was observed.

Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL and 10 µg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 µg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 µg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 µg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

Developing a Chromochloris zofingiensis Mutant for Enhanced Production of Lutein under CO2 Aeration.

Microalgae are competitive and commercial sources for health-benefit carotenoids. In this study, a Chromochloris zofingiensis mutant (Cz-pkg), which does not shut off its photosystem and stays green upon glucose treatment, was generated and characterized. Cz-pkg was developed by treating the algal cells with a chemical mutagen as N-methyl-N'-nitro-N-nitrosoguanidine and followed by a color-based colony screening approach. Cz-pkg was found to contain a dysfunctional cGMP-dependent protein kinase (PKG). By cultivated with CO2 aeration under mixotrophy, the mutant accumulated lutein up to 31.93 ± 1.91 mg L-1 with a productivity of 10.57 ± 0.73 mg L-1 day-1, which were about 2.5- and 8.5-fold of its mother strain. Besides, the lutein content of Cz-pkg could reach 7.73 ± 0.52 mg g-1 of dry weight, which is much higher than that of marigold flower, the most common commercial source of lutein. Transcriptomic analysis revealed that in the mutant Cz-pkg, most of the genes involved in the biosynthesis of lutein and chlorophylls were not down-regulated upon glucose addition, suggesting that PKG may regulate the metabolisms of photosynthetic pigments. This study demonstrated that Cz-pkg could serve as a promising strain for both lutein production and glucose sensing study.

Ethylene-induced potassium transporter AcKUP2 gene is involved in kiwifruit postharvest ripening.

BACKGROUND: Potassium (K) is important in the regulation of plant growth and development. It is the most abundant mineral element in kiwifruit, and its content increases during fruit ripening. However, how K+ transporter works in kiwifruit postharvest maturation is not yet clear. RESULTS: Here, 12 K+ transporter KT/HAK/KUP genes, AcKUP1 ~ AcKUP12, were isolated from kiwifruit, and their phylogeny, genomic structure, chromosomal location, protein properties, conserved motifs and cis-acting elements were analysed. Transcription analysis revealed that AcKUP2 expression increased rapidly and was maintained at a high level during postharvest maturation, consistent with the trend of K content; AcKUP2 expression was induced by ethylene, suggesting that AcKUP2 might play a role in ripening. Fluorescence microscopy showed that AcKUP2 is localised in the plasma membrane. Cis-elements, including DER or ethylene response element (ERE) responsive to ethylene, were found in the AcKUP2 promoter sequence, and ethylene significantly enhanced the AcKUP2 promoter activity. Furthermore, we verified that AcERF15, an ethylene response factor, directly binds to the AcKUP2 promoter to promote its expression. Thus, AcKUP2 may be an important potassium transporter gene which involved in ethylene-regulated kiwifruit postharvest ripening. CONCLUSIONS: Therefore, our study establishes the first genome-wide analysis of the kiwifruit KT/HAK/KUP gene family and provides valuable information for understanding the function of the KT/HAK/KUP genes in kiwifruit postharvest ripening.

KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana.

SignificanceKarrikins are chemicals in smoke that stimulate regrowth of many plants after fire. However, karrikin responses are not limited to species from fire-prone environments and can affect growth after germination. Putatively, this is because karrikins mimic an unknown signal in plants, KAI2 ligand (KL). Karrikins likely require modification in plants to become bioactive. We identify a gene, KUF1, that appears to negatively regulate biosynthesis of KL and metabolism of a specific karrikin. KUF1 expression increases in response to karrikin or KL signaling, thus forming a negative feedback loop that limits further activation of the signaling pathway. This discovery will advance understanding of how karrikins are perceived and how smoke-activated germination evolved. It will also aid identification of the elusive KL.

Camellia sinensis small GTPase gene (CsRAC1) involves in response to salt stress, drought stress and ABA signaling pathway.

RAC/ROP gene (RACs) is a plant-specific small GTPases. RACs play an irreplaceable role in the tissue dynamics of cytoskeleton, vesicle transport and hormone signal transmission in plants. In the present study, a novel gene from RACs family, CsRAC1, was identified from tea [Camellia sinensis (L.) O. Kuntze]. CsRAC1 contained a 591-bp open reading frame and encoded a putative protein of 197 amino acids. Subcellular localization analysis in leaves of transgenic tobacco and root tips of Arabidopsis thaliana showed that CsRAC1 targeted the nucleus and cell membrane. The expression of CsRAC1 induced by abiotic stresses such as cold, heat, drought, salt and abscisic acid has also been verified by RT-qPCR. Further verification of biological function of CsRAC1 showed that overexpression of CsRAC1 increased the sensitivity of A. thaliana to salt stress, improved the tolerance of mature A. thaliana to drought stress, and enhanced the inhibition of ABA on seed germination of A. thaliana. In addition, the antioxidant system regulated by CsRAC1 mainly worked in mature A. thaliana. The results indicate that CsRAC1 is involved in the response of C. sinensis to salt, drought stress and ABA signaling pathway.

Exogenously-Sourced Ethylene Positively Modulates Photosynthesis, Carbohydrate Metabolism, and Antioxidant Defense to Enhance Heat Tolerance in Rice.

The effect of exogenously-applied ethylene sourced from ethephon (2-chloroethyl phosphonic acid)was studied on photosynthesis, carbohydrate metabolism, and high-temperature stress tolerance in Taipei-309 and Rasi cultivars of rice (Oryza sativa L.). Heat stress increased the content of H2O2 and thiobarbituric acid reactive substances (TBARS)more in Rasi than Taipei-309. Further, a significant decline in sucrose, starch, and carbohydrate metabolism enzyme activity and photosynthesis was also observed in response to heat stress. The application of ethephon reduced H2O2 and TBARS content by enhancing the enzymatic antioxidant defense system and improved carbohydrate metabolism, photosynthesis, and growth more conspicuously in Taipei-309 under heat stress. The ethephon application enhanced photosynthesis by up-regulating the psbA and psbB genes of photosystem II in heat-stressed plants. Interestingly, foliar application of ethephoneffectively down-regulated high-temperature-stress-induced elevated ethylene biosynthesis gene expression. Overall, ethephon application optimized ethylene levels under high-temperature stress to regulate the antioxidant enzymatic system and carbohydrate metabolism, reducing the adverse effects on photosynthesis. These findings suggest that ethylene regulates photosynthesis via carbohydrate metabolism and the antioxidant system, thereby influencing high-temperature stress tolerance in rice.

Genome-Wide Identification and Characterization of the Soybean DEAD-Box Gene Family and Expression Response to Rhizobia.

DEAD-box proteins are a large family of RNA helicases that play important roles in almost all cellular RNA processes in model plants. However, little is known about this family of proteins in crops such as soybean. Here, we identified 80 DEAD-box family genes in the Glycine max (soybean) genome. These DEAD-box genes were distributed on 19 chromosomes, and some genes were clustered together. The majority of DEAD-box family proteins were highly conserved in Arabidopsis and soybean, but Glyma.08G231300 and Glyma.14G115100 were specific to soybean. The promoters of these DEAD-box genes share cis-acting elements involved in plant responses to MeJA, salicylic acid (SA), low temperature and biotic as well as abiotic stresses; interestingly, half of the genes contain nodulation-related cis elements in their promoters. Microarray data analysis revealed that the DEAD-box genes were differentially expressed in the root and nodule. Notably, 31 genes were induced by rhizobia and/or were highly expressed in the nodule. Real-time quantitative PCR analysis validated the expression patterns of some DEAD-box genes, and among them, Glyma.08G231300 and Glyma.14G115100 were induced by rhizobia in root hair. Thus, we provide a comprehensive view of the DEAD-box family genes in soybean and highlight the crucial role of these genes in symbiotic nodulation.

Salicylic Acid Enhances Adventitious Root and Aerenchyma Formation in Wheat under Waterlogged Conditions.

The present study investigated the role of salicylic acid (SA) in regulating morpho-anatomical adaptive responses of a wheat plant to waterlogging. Our pharmacological study showed that treatment of waterlogged wheat plants with exogenous SA promotes the formation axile roots and surface adventitious roots that originate from basal stem nodes, but inhibits their elongation, leading to the formation of a shallow root system. The treatment also enhanced axile root formation in non-waterlogged plants but with only slight reductions in their length and branch root formation. Exogenous SA enhanced the formation of root aerenchyma, a key anatomical adaptive response of plants to waterlogging. Consistent with these results, waterlogging enhanced SA content in the root via expression of specific isochorismate synthase (ICS; ICS1 and ICS2) and phenylalanine ammonia lyase (PAL; PAL4, PAL5 and PAL6) genes and in the stem nodes via expression of specific PAL (PAL5 and PAL6) genes. Although not to the same level observed in waterlogged plants, exogenous SA also induced aerenchyma formation in non-waterlogged plants. The findings of this study furthermore indicated that inhibition of ethylene synthesis in SA treated non-waterlogged and waterlogged plants does not have any effect on SA-induced emergence of axile and/or surface adventitious roots but represses SA-mediated induction of aerenchyma formation. These results highlight that the role of SA in promoting the development of axile and surface adventitious roots in waterlogged wheat plants is ethylene independent while the induction of aerenchyma formation by SA requires the presence of ethylene.

Salicylic Acid Is Required for Broad-Spectrum Disease Resistance in Rice.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.

Hydrogen Sulfide-Linked Persulfidation Maintains Protein Stability of ABSCISIC ACID-INSENSITIVE 4 and Delays Seed Germination.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays an important role in the plant life cycle. The multiple transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) was precisely regulated to participate in the abscisic acid (ABA) mediated signaling cascade. However, the molecular mechanisms of how H2S regulates ABI4 protein level to control seed germination and seedling growth have remained elusive. In this study, we demonstrated that ABI4 controls the expression of L-CYSTEINE DESULFHYDRASE1 (DES1), a critical endogenous H2S-producing enzyme, and both ABI4 and DES1-produced H2S have inhibitory effects on seed germination. Furthermore, the ABI4 level decreased during seed germination while H2S triggered the enhancement of the persulfidation level of ABI4 and alleviated its degradation rate, which in turn inhibited seed germination and seedling establishment. Conversely, the mutation of ABI4 at Cys250 decreased ABI4 protein stability and facilitated seed germination. Moreover, ABI4 degradation is also regulated via the 26S proteasome pathway. Taken together, these findings suggest a molecular link between DES1 and ABI4 through the post-translational modifications of persulfidation during early seedling development.

Nitrogen Uptake and Distribution in Different Chinese Cabbage Genotypes under Low Nitrogen Stress.

In order to understand the effects of low nitrogen (LN) stress on the growth and development in different genotypes of Chinese cabbage, the L40 genotype with high nitrogen utilization and the L14 genotype with LN utilization were selected as experimental materials. Field experiments and indoor hydroponic methods were used to study the different responses of two Chinese cabbage genotypes to low nitrogen levels. In this study, we also analyzed the genome-wide gene expression profiles of L40 and L14 in response to LN stress by high-throughput RNA sequencing technology. The results reveal that the L40 root system responds better to LN compared with L14. After LN stress, L40 can effectively absorb and transport NO3- and store it in the ground. It is precisely because of this characteristic of the L40 genotype that LN treatment did not have a significant effect on the chlorophyll (Chl) content and net photosynthetic rate (Pn) of the L40 Chinese cabbage compared with the L14 Chinese cabbage. These two different Chinese cabbage genotypes were shown to have differently expressed genes related to nitrate transport, auxin synthesis, and glutamate dehydrogenase synthesis. These genes function in the nitrogen pathway, which are important candidates for understanding the molecular host-response mechanisms to LN stress.

Nitric Oxide Extends the Postharvest Life of Water Bamboo Shoots Partly by Maintaining Mitochondrial Structure and Energy Metabolism.

Harvested water bamboo shoots can be stored for only a few days before they lose weight and become soft. Nitrogen oxide (NO) and modified atmosphere packaging (MAP) have previously been used to prolong horticultural crop storage. In the present study, we analyzed the joint effect of these two methods on extending the postharvest quality of water bamboo shoots. Water bamboo shoots were treated with (1) 30 µL L-1 NO, (2) MAP, and (3) a combination of NO and MAP. The NO treatment delayed the softness and weight loss through maintaining the integrity of the mitochondrial ultrastructure and enhancing the ATP level by activating the expressions and activities of succinic dehydrogenase, malic acid dehydrogenase, and cytochrome oxidase. MAP improved the effect of NO on the mitochondrial energy metabolism. These results indicate that NO and MAP treatments are effective at suppressing the quality deterioration of water bamboo shoots, MAP improves the effect of NO in extending postharvest life, and NO may be the main effective factor in the combination of NO and MAP.

Genome-wide identification, expression and functional analysis of the core cell cycle gene family during the early somatic embryogenesis of Dimocarpus longan Lour.

Core cell cycle genes (CCCs) are essential regulators of cell cycle operation. In this study, a total of 69 CCCs family members, including 37 CYCs, 20 CDKs, five E2F/DPs, three KRPs, two RBs, one CKS and one Wee1, were identified from the longan genome. Phylogenetic and motifs analysis showed the evolutionary conservation of CCCs. Transcriptome dataset showed that CCCs had various expression patterns during longan early somatic embryogenesis (SE). Either CKS or CYCD3;2 silencing increased the expression of RB-E2F pathway genes, and the silencing of CYCD3;2 might induce the process of apoptosis in longan embryogenic callus (EC) cells. In addition, The qRT-PCR results showed that the expression levels of CDKG2, CYCD3;2, CYCT1;2, CKS and KRP1 were elevated by ABA, 2,4-D and PEG4000 treatments, while CDKG2 and CYCT1;2 were inhibited by NaCl treatment. In conclusion, our study provided valuable information for understanding the characterization and biological functions of longan CCCs.

Genome-wide identification and expression analysis response to GA3 stresses of WRKY gene family in seed hemp (Cannabis sativa L).

WRKY transcription factor is one of the largest transcription factor families in higher plants. However, the investigations of the WRKY gene family have not yet been reported in seed hemp. In the present study, we identified 39 CasWRKYs at the genome-wide level and analyzed phylogenetic relationship, chromosome location, cis-acting elements, gene structure, conserved motif, and expression pattern. Based on the gene structure and phylogenetic analyses, CasWRKY proteins were divided into 3 groups and 7 subgroups. The gene duplication investigation revealed that 6 and 5 pairs of CasWRKY genes underwent tandem and segmental duplication events, respectively. These events may contribute to the diversity and expansion of the CasWRKY gene family. The regulatory elements in the promoter regions of CasWRKYs contained diverse cis-regulatory elements, among which P-box cis-regulatory elements showed high frequency, indicating that CasWRKYs can respond to the regulation of gibberellin. The expression profiles derived from RNA-seq and qRT-PCR showed that 13 CasWRKY genes could respond to GA3 stress and affect fiber development, as well as play significant roles in stem growth and development. This study will serve as molecular basis and practical reference for further exploring the genetic evolution and biological function of CasWRKY genes in seed hemp.

Metabolic stimulation-elicited transcriptional responses and biosynthesis of acylated triterpenoids precursors in the medicinal plant Helicteres angustifolia.

BACKGROUND: Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation. RESULTS: Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively. CONCLUSION: In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and ß-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis.