Biotechnological approaches for the production of camptothecin.

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved . KEY POINTS: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production.

Pyramiding of transcription factor, PgHSF4, and stress-responsive genes of p68, Pg47, and PsAKR1 impart multiple abiotic stress tolerance in rice (Oryza sativa L.).

Abiotic stresses such as drought, salinity, and heat stress significantly affect rice crop growth and production. Under uncertain climatic conditions, the concurrent multiple abiotic stresses at different stages of rice production became a major challenge for agriculture. Hence, improving rice's multiple abiotic stress tolerance is essential to overcome unprecedented challenges under adverse environmental conditions. A significant challenge for rice breeding programs in improving abiotic stress tolerance involves multiple traits and their complexity. Multiple traits must be targeted to improve multiple stress tolerance in rice and uncover the mechanisms. With this hypothesis, in the present study gene stacking approach is used to integrate multiple traits involved in stress tolerance. The multigene transgenics co-expressing Pennisetum glaucum 47 (Pg47), Pea 68 (p68), Pennisetum glaucum Heat Shock Factor 4(PgHSF4), and Pseudomonas Aldo Keto Reductase 1 (PsAKR1) genes in the rice genotype (AC39020) were developed using the in-planta transformation method. The promising transgenic lines maintained higher yields under semi-irrigated aerobic cultivation (moisture stress). These 15 promising transgenic rice seedlings showed improved shoot and root growth traits under salinity, accelerating aging, temperature, and oxidative stress. They showed better physiological characteristics, such as chlorophyll content, membrane stability, and lower accumulation of reactive oxygen species, under multiple abiotic stresses than wild-type. Enhanced expression of transgenes and other stress-responsive downstream genes such as HSP70, SOD, APX, SOS, PP2C, and P5CS in transgenic lines suggest the possible molecular mechanism for imparting the abiotic stress tolerance. This study proved that multiple genes stacking as a novel strategy induce several mechanisms and responsible traits to overcome multiple abiotic stresses. This multigene combination can potentially improve tolerance to multiple abiotic stress conditions and pave the way for developing climate-resilient crops.

Transcriptome profiling, physiological, and biochemical analyses provide new insights towards drought stress response in sugar maple (Acer saccharum Marshall) saplings.

Sugar maple (Acer saccharum Marshall) is a temperate tree species in the northeastern parts of the United States and is economically important for its hardwood and syrup production. Sugar maple trees are highly vulnerable to changing climatic conditions, especially drought, so understanding the physiological, biochemical, and molecular responses is critical. The sugar maple saplings were subjected to drought stress for 7, 14, and 21 days and physiological data collected at 7, 14, and 21 days after stress (DAS) showed significantly reduced chlorophyll and Normalized Difference Vegetation Index with increasing drought stress time. The drought stress-induced biochemical changes revealed a higher accumulation of malondialdehyde, proline, and peroxidase activity in response to drought stress. Transcriptome analysis identified a total of 14,099 differentially expressed genes (DEGs); 328 were common among all stress periods. Among the DEGs, transcription factors (including NAC, HSF, ZFPs, GRFs, and ERF), chloroplast-related and stress-responsive genes such as peroxidases, membrane transporters, kinases, and protein detoxifiers were predominant. GO enrichment and KEGG pathway analysis revealed significantly enriched processes related to protein phosphorylation, transmembrane transport, nucleic acids, and metabolic, secondary metabolite biosynthesis pathways, circadian rhythm-plant, and carotenoid biosynthesis in response to drought stress. Time-series transcriptomic analysis revealed changes in gene regulation patterns in eight different clusters, and pathway analysis by individual clusters revealed a hub of stress-responsive pathways. In addition, qRT-PCR validation of selected DEGs revealed that the expression patterns were consistent with transcriptome analysis. The results from this study provide insights into the dynamics of physiological, biochemical, and gene responses to progressive drought stress and reveal the important stress-adaptive mechanisms of sugar maple saplings in response to drought stress.

Genomic and evolutionary relationships among wild and cultivated blueberry species.

BACKGROUND: Blueberries (Vaccinium section Cyanococcus) are an economically important fruit crop in the United States. Understanding genetic structure and relationships in blueberries is essential to advance the genetic improvement of horticulturally important traits. In the present study, we investigated the genomic and evolutionary relationships in 195 blueberry accessions from five species (comprising 33 V. corymbosum, 14 V. boreale, 81 V. darrowii, 29 V. myrsinites, and 38 V. tenellum) using single nucleotide polymorphisms (SNPs) mined from genotyping-by-sequencing (GBS) data. RESULTS: GBS generated ~ 751 million raw reads, of which 79.7% were mapped to the reference genome V. corymbosum cv. Draper v1.0. After filtering (read depth > 3, minor allele frequency > 0.05, and call rate > 0.9), 60,518 SNPs were identified and used in further analyses. The 195 blueberry accessions formed three major clusters on the principal component (PC) analysis plot, in which the first two PCs accounted for 29.2% of the total genetic variance. Nucleotide diversity (π) was highest for V. tenellum and V. boreale (0.023 each), and lowest for V. darrowii (0.012). Using TreeMix analysis, we identified four migration events and deciphered gene flow among the selected species. In addition, we detected a strong V. boreale lineage in cultivated blueberry species. Pairwise SweeD analysis identified a wide sweep (encompassing 32 genes) as a strong signature of domestication on the scaffold VaccDscaff 12. From this region, five genes encoded topoisomerases, six genes encoded CAP-gly domain linker (which regulates the dynamics of the microtubule cytoskeleton), and three genes coded for GSL8 (involved in the synthesis of the cell wall component callose). One of the genes, augustus_masked-VaccDscaff12-processed-gene-172.10, is a homolog of Arabidopsis AT2G25010 and encodes the protein MAINTENANCE OF MERISTEMS-like involved in root and shoot growth. Additional genomic stratification by admixture analysis identified genetic lineages and species boundaries in blueberry accessions. The results from this study indicate that V. boreale is a genetically distant outgroup, while V. darrowii, V. myrsinites, and V. tenellum are closely related. CONCLUSION: Our study provides new insights into the evolution and genetic architecture of cultivated blueberries.

Stacking herbicide detoxification and resistant genes improves glyphosate tolerance and reduces phytotoxicity in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.).

Glyphosate residues retained in the growing meristematic tissues or in grains of glyphosate-resistant crops affect the plants physiological functions and crop yield. Removing glyphosate residues in the plants is desirable with no penalty on crop yield and quality. We report a new combination of scientific strategy to detoxify glyphosate that reduces the residual levels and improve crop resistance. The glyphosate detoxifying enzymes Aldo-keto reductase (AKR1) and mutated glycine oxidase (mGO) with different modes of action were co-expressed with modified EPSPS, which is insensitive to glyphosate in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.). The transgenic tobacco plants expressing individual PsAKR1, mGO, CP4-EPSPS, combinations of PsAKR1:CP4EPSPS, PsAKR1:mGO, and multigene with PsAKR1: mGO: CP4EPSPS genes were developed. The bio-efficacy studies of in-vitro leaf regeneration on different concentrations of glyphosate, seedling bioassay, and spray on transgenic tobacco plants demonstrate that glyphosate detoxification with enhanced resistance. Comparative analysis of the transgenic tobacco plants reveals that double and multigene expressing transgenics had reduced accumulation of shikimic acid, glyphosate, and its primary residue AMPA, and increased levels of sarcosine were observed in all PsAKR1 expressing transgenics. The multigene expressing rice transgenics showed improved glyphosate resistance with yield maintenance. In summary, results suggest that stacking genes with two different detoxification mechanisms and insensitive EPSPS is a potential approach for developing glyphosate-resistant plants with less residual content.

Differential Morpho-Physiological and Transcriptomic Responses to Heat Stress in Two Blueberry Species.

Blueberries (Vaccinium spp.) are highly vulnerable to changing climatic conditions, especially increasing temperatures. To gain insight into mechanisms underpinning the response to heat stress, two blueberry species were subjected to heat stress for 6 and 9 h at 45 °C, and leaf samples were used to study the morpho-physiological and transcriptomic changes. As compared with Vaccinium corymbosum, Vaccinium darrowii exhibited thermal stress adaptation features such as small leaf size, parallel leaf orientation, waxy leaf coating, increased stomatal surface area, and stomatal closure. RNAseq analysis yielded ~135 million reads and identified 8305 differentially expressed genes (DEGs) during heat stress against the control samples. In V. corymbosum, 2861 and 4565 genes were differentially expressed at 6 and 9 h of heat stress, whereas in V. darrowii, 2516 and 3072 DEGs were differentially expressed at 6 and 9 h, respectively. Among the pathways, the protein processing in the endoplasmic reticulum (ER) was the highly enriched pathway in both the species: however, certain metabolic, fatty acid, photosynthesis-related, peroxisomal, and circadian rhythm pathways were enriched differently among the species. KEGG enrichment analysis of the DEGs revealed important biosynthesis and metabolic pathways crucial in response to heat stress. The GO terms enriched in both the species under heat stress were similar, but more DEGs were enriched for GO terms in V. darrowii than the V. corymbosum. Together, these results elucidate the differential response of morpho-physiological and molecular mechanisms used by both the blueberry species under heat stress, and help in understanding the complex mechanisms involved in heat stress tolerance.

Admixture Analysis Using Genotyping-by-Sequencing Reveals Genetic Relatedness and Parental Lineage Distribution in Highbush Blueberry Genotypes and Cross Derivatives.

Blueberries (Vaccinium section Cyanococcus) are perennial shrubs widely cultivated for their edible fruits. In this study, we performed admixture and genetic relatedness analysis of northern highbush (NHB, primarily V. corymbosum) and southern highbush (SHB, V. corymbosum introgressed with V. darrowii, V. virgatum, or V. tenellum) blueberry genotypes, and progenies of the BNJ16-5 cross (V. corymbosum × V. darrowii). Using genotyping-by-sequencing (GBS), we generated more than 334 million reads (75 bp). The GBS reads were aligned to the V. corymbosum cv. Draper v1.0 reference genome sequence, and ~2.8 million reads were successfully mapped. From the alignments, we identified 2,244,039 single-nucleotide polymorphisms, which were used for principal component, haplotype, and admixture analysis. Principal component analysis revealed three main groups: (1) NHB cultivars, (2) SHB cultivars, and (3) BNJ16-5 progenies. The overall fixation index (FST) and nucleotide diversity for NHB and SHB cultivars indicated wide genetic differentiation, and haplotype analysis revealed that SHB cultivars are more genetically diverse than NHB cultivars. The admixture analysis identified a mixture of various lineages of parental genomic introgression. This study demonstrated the effectiveness of GBS-derived single-nucleotide polymorphism markers in genetic and admixture analyses to reveal genetic relatedness and to examine parental lineages in blueberry, which may be useful for future breeding plans.

Characterization and Expression Analysis of Common Bean Histone Deacetylase 6 during Development and Cold Stress Response.

Histone deacetylases (HDACs) are important regulators of gene transcription thus controlling multiple cellular processes. Despite its essential role in plants, HDA6 is yet to be validated in common bean. In this study, we show that HDA6 is involved in plant development and stress response. Differential expression of HDA6 was determined in various tissues and the expression was seen to be upregulated with plant age (seedling < flowering < maturity). Higher expression was observed in flowers and pods than in stem, leaf, and root. Upregulation of HDA6 gene during cold stress implies its prominent role in abiotic stress. Furthermore, the HDA6 gene was isolated from three common bean genotypes and sequence analyses revealed homology with functionally characterized homologs in model species. The 53 kDa translated product was detected using an HDA6 specific antibody and recombinant protein overexpressed in Escherichia coli showed HDAC activity in vitro. To our knowledge, this is the first report in the agriculturally important crop common bean describing the functional characterization and biological role of HDA6.

Transcriptional Analysis of Resistance to Low Temperatures in Bermudagrass Crown Tissues.

Bermudagrass (Cynodon dactylon L pers.) is one of the most geographically adapted and utilized of the warm-season grasses. However, bermudagrass adaptation to the Northern USA is limited by freeze damage and winterkill. Our study provides the first large-scale analyses of gene expression in bermudagrass regenerative crown tissues during cold acclimation. We compared gene expression patterns in crown tissues from highly cold tolerant "MSU" and susceptible "Zebra" genotypes exposed to near-freezing temperatures. Suppressive subtractive hybridization was used to isolate putative cold responsive genes Approximately, 3845 transcript sequences enriched for cold acclimation were deposited in the GenBank. A total of 4589 ESTs (3184 unigenes) including 744 ESTs associated with the bermudagrass disease spring dead spot were printed on microarrays and hybridized with cold acclimated complementary Deoxyribonucleic acid (cDNA). A total of 587 differentially expressed unigenes were identified in this study. Of these only 97 (17%) showed significant NCBI matches. The overall expression pattern revealed 40% more down- than up-regulated genes, which was particularly enhanced in MSU compared to Zebra. Among the up-regulated genes 68% were uniquely expressed in MSU (36%) or Zebra (32%). Among the down-regulated genes 40% were unique to MSU, while only 15% to Zebra. Overall expression intensity was significantly higher in MSU than in Zebra (p value ≤ 0.001) and the overall number of genes expressed at 28 days was 2.7 fold greater than at 2 days. These changes in expression patterns reflect the strong genotypic and temporal response to cold temperatures. Additionally, differentially expressed genes from this study can be utilized for developing molecular markers in bermudagrass and other warm season grasses for enhancing cold hardiness.

Quantification and Gene Expression Analysis of Histone Deacetylases in Common Bean during Rust Fungal Inoculation.

Histone deacetylases (HDACs) play an important role in plant growth, development, and defense processes and are one of the primary causes of epigenetic modifications in a genome. There was only one study reported on epigenetic modifications of the important legume crop, common bean, and its interaction with the fungal rust pathogen Uromyces appendiculatus prior to this project. We measured the total active HDACs levels in leaf tissues and observed expression patterns for the selected HDAC genes at 0, 12, and 84 hours after inoculation in mock inoculated and inoculated plants. Colorimetric analysis showed that the total amount of HDACs present in the leaf tissue decreased at 12 hours in inoculated plants compared to mock inoculated control plants. Gene expression analyses indicated that the expression pattern of gene PvSRT1 is similar to the trend of total active HDACs in this time course experiment. Gene PvHDA6 showed increased expression in the inoculated plants during the time points measured. This is one of the first attempts to study expression levels of HDACs in economically important legumes in the context of plant pathogen interactions. Findings from our study will be helpful to understand trends of total active HDACs and expression patterns of these genes under study during biotic stress.

Identification and analysis of common bean (Phaseolus vulgaris L.) transcriptomes by massively parallel pyrosequencing.

BACKGROUND: Common bean (Phaseolus vulgaris) is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. RESULTS: We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt). These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC) end sequences, and a total of 21% of the unigenes (12,724) including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs) and transcription factors were also identified in this study. CONCLUSIONS: This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and will aid in the development of molecular markers that can be used for tagging genes of interest. Additionally, these sequences will provide a means for better annotation of the on-going common bean whole genome sequencing.

Drought response in the spikes of barley: gene expression in the lemma, palea, awn, and seed.

The photosynthetic organs of the barley spike (lemma, palea, and awn) are considered resistant to drought. However, there is little information about gene expression in the spike organs under drought conditions. We compared response of the transcriptome of the lemma, palea, awn, and seed to drought stress using the Barley1 Genome Array. Barley plants were exposed to drought treatment for 4 days at the grain-filling stage by withholding water. At the end of the stress, relative water content of the lemma, palea, and awn dropped from 85% to 60%. Nevertheless, the water content of the seed only decreased from 89% to 81%. Transcript abundance followed the water status of the spike organs; the awn had more drought-regulated genes followed by lemma and palea, and the seed showed very little change in gene expression. Despite expressing more drought-associated genes, many genes for amino acid, amino acid derivative, and carbohydrate metabolism, as well as for photosynthesis, respiration, and stress response, were down-regulated in the awn compared with the lemma, palea, and seed. This suggests that the lemma and the palea are more resistant to drought stress compared with the awn.