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336 S. Pattnaik
Table 18.1 The list of stress-related genes, provoking plant to express phytoconstituents under stress, using secondary metabolism pathway
Sl
no. Names of genes associated with stress Type of stress Plant
Metabolic product with
medicinal importance References
1 Osmoregulation (Δ1P5CS), detoxification (GST
and SOD), signal transduction (STK and PSP),
metabolism (AD and LD), and transcription
regulation (HSP, MYB, and WRKY) were
considered to show their enhanced responses
under drought stress condition
Drought Withania somnifera Sanchita and
Sharma
(2018)
2 RghBNG gene Abiotic stress Rehmannia glutinosa
3 Amorpha-4,11-diene synthase (ADS) and
cytochrome P450 monooxygenase
(CYP71AV1) genes
Chilling, heat shock,
or ultraviolet
(UV) light,
Artemisia annua Terpenoid indole alkaloids
UV-B Catharanthus roseus
4 Panax quinquefolius Ginsenoside
5 l C-4 methyl oxidase gene (SMO1 gene Dehydration/drought
stress
Artemisia annua Sterol
6 AP2/ERF TF gene Wounding Papaver somniferum Benzylisoquinoline
alkaloids, narcotine and
papaverine
7 Actin1 (ACT), clathrin complex AP1 (CAC),
cyclophilin (CYP), heat-shock protein 40
(DNAJ), dehydration responsive element
binding gene (DREB), translation elongation
factor1 (EF1), Ras-related protein (RAN),
translation initiation factor (TIF1), βTubulin
(TUB), ubiquitin-conjugating enzyme E2
(UBC2), ATP binding-box transporter
2 (ABCC2), COP9 signal complex subunit 3
(COPS3), citrate synthase (CS), and R3H
domain
protein 2 (R3HDM2)
Osmotic and salinity Glycyrrhiza uralensis
F. and Glycyrrhiza
inflata B.
Glycyrrhizin
Li et al.
(
2020)
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18 Next-Generation Sequencing (NGS) for Metabolomics Study in... 337
Fig. 18.9 The key steps accommodated in WGS approaches. (a) the plant exposed with stress
factors like drought, salinity; (b) extraction of total RNA; (c) gating of mRNA; (d) making cDNA;
(e) sequencing and aligning contigs; (f) filling the gap and making a contiguous stretch of DNA; (g)
visualizing the sequences through pertinent bioinformatics software
Fig. 18.10 A typical transcriptomics score board. The analysis of POIUS of medicinal plants
showing the degree of gene expression, as obtained from real time transcription, annotated with
gene functions, GO (gene ontology) terms and KEGG pathway
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338 S. Pattnaik
18.1.6 WGS of Genomes of Representative Medicinal and Aromatic
Plant Species with Defined Antimicrobial Activity
There are several medicinal and aromatic plants, are defined with remarkable
antimicrobial activity, which were reported earlier, as author’s research, are consid-
ered as treasure trove of plant-derived pharmaceutical s. The essential oils and its
phytoconstituents belonging to category of terpenoids, sesquiterpenoids,
monoterpenes, etc. have occupied a designated place in pharma industry. In the
past and also in the future, efforts are made to carry out WGS, of respective
genomes, to detect the genes associated with POIUS.
Among the plant species, Eucalyptus sp., belonging to family Myrtle
(Myrtaceae), has been source of medicine since time immemorial. Genus Eucalyptus
is considered as a major source 1,8-cineol (49.07–83.59%) and α-pinene
(1.27–26.35%), which are components of Eucalyptus essential oil, having enough
medicinal importance. Therefore, to amplify the Eucalyptus active constituents,
investigators had taken interest and opted for WGS of the specific plant species.
Eucalyptus tereticornis (ET217 and ET86) and one each from E. camaldulensis
(EC17) and E. grandis (EG9) were subjected to whole-genome resequencing.
Low-coverage (10×) genome sequencing was used to find polymorphic SSRs
between the individuals (Patturaj et al.
2021). The average number of SSR loci
identified was 95,513, and the density of SSRs per Mb was from 157.39 in EG9 to
155.08 in EC17. Among all the SSRs detected, the most abundant repeat motifs were
dinucleotide (59.6–62.5%), followed by tri- (23.7–27.2%), tetra- (5.2–5.6%), penta-
(5.0–5.3%), and hexa-nucleotide (2.7–2.9%). The predominant SSR motif units
were AG/CT and AAG/TTC. The computational genome analysis had predicted
the SSR length variations between the individuals and identified the gene functions
of SSR-containing sequences. The markers, for single-nucleotide polymorphisms
(SNP), InDels, and transcriptional regulators were also predicted by the authors
using omics tools and techniques. Previously, (Yaradua and Shah 2020) there was
identification of 172 loci in the Eucalyptus grandis genome with a high sequence
similarity to known terpene synthase genes from other species. Genome sequences
of Eucalyptus grandis and Eucalyptus globulus were mined for terpene synthase
genes (TPS) (Karunanithi and Zerbe
2019), while Euc alyptus grandis nonredundant
chromosome-scale reference (V1.0) sequence for BRASUZ1 based on 6.7× whole-
genome Sanger shotgun coverage, paired bacterial artificial chromosome (BAC)-end
sequencing, and a high-density genetic linkage map was established (Myburg et al.
2014). An estimated 94% of the genome is organized into 11 pseudomolecules
(605 megabases (Mb)). Anchoring the genome assembly to an independent linkage
map 7 revealed that the remaining 4941 smaller unanchored scaffolds (totalling
85 Mb) correspond largely to repeat-rich sequences. Eucalyptus also shows the
highest diversity of genes for specialized metabolites such as terpenes that act as
chemical defense and provide unique pharmaceutical oils. Heat shock transcription
factors (HSFs) activate heat shock protein gene expression by binding their
promoters in response to heat stress and are considered to be pivotal transcription
factors in plants (Yuan et a l.
2022). Eucalyptus is a superior source of fuel and
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commercial wood. During its growth, high temperature or other abiotic stresses
could impact its defense capability and growth. Hsf genes have been cloned and
sequenced in many plants but rarely in Eucalyptus. In this study, the authors had
used bioinformatics methods to analyze and identify Eucalyptus Hsf genes, their
chromosomal localization, and structure. The phylogenetic relations hip and
conserved domains of their encoded proteins were further analyzed. A tot
al of
36 Hsf genes wer e identified and authenticated from Eucalyptus, which were
scattered across 11 chromosomes. They could be classified into three classes
(A, B, and C). Additionally, a large number of stress-related cis-regulatory elements
were identified in the upstream promoter sequence of HSF, and cis-acting element
analysis indicated that the expression of EgHsf may be regulated by plant growth and
development, metabolism, hormones, and stress responses. The r
epresentative five
Hsf genes, EgHsf4, EgHsf9, EgHsf13, EgHsf24, and EgHsf32, under salt and
temperature stresses are amplified by qRT-PCR. The expressions of all tested Hsf
genes in six tissues were at different levels. Finally, we investigated the network of
interplay between genes, and the results suggest that there may be synergistic effects
between different Hsf genes in response to abiotic stresses.
18 Next-Generation Sequencing (NGS) for Metabolomics Study in... 339
Likew
ise, Ment
ha spp.
a
re also considered by some authors, because of its
equivalent medicinal properties with Eucalyptus genera. The WGS was carried out
for analyzing the TPS (terpene synthase) gene family (Chen et al. 2021) and in
Mentha longifolia, a model plant for functional genomic research in the genus
Mentha. A total of 63 TPS genes were being identified in the M. longifolia genome
sequence assembly, which could be divided into six subfamilies. The TPS-b sub-
family had the largest number of genes, which might be related to the abundant
monoterpenoids in Mentha plants. The TPS-e subfamily has 18 members. The
63 TPS genes could be mapped to 9 scaffolds of the M. longifolia genome sequence
assembly, and the distribution of these genes is uneven. Tandem duplicates and
fragment duplicates contributed greatly to the increase in the number of TPS genes in
M. longifolia. The conserved motifs (RR(X) 8W, NSE/DTE, RXR, and DDXXD)
were analyzed in M. longifolia TPSs, and significant differentiation was found
between different subfamilies. In addition, the authors could clone and analyze
the catalytic activity of a single terpene synthase, MlongTPS29, which belongs to
the TPS-b subfamily. MlongTPS29 could encode a limonene synthase and catalyze
the biosynthesis of limonene, an important precursor of essential oils from the genus
Mentha.
There was de novo transc riptomics analysis of L. camara L. (Asteraceae)
(Pattnaik and Pattnaik 2010; Pradhan and Pattnaik 2017) leaf and root revealing
70,155,594 and 84,263,224 clean reads with elution of 72,877 and 513,985 unigenes
from leaf and root, respectively (Shah et al.
2020). The pathway analysis had
inferred about the presence of 229 and 943 genes involved in the phenylpropanoid
biosynthesis in leaf and root tissues, respectively. The complete chloroplast genome
of said plant, being an important medicinal plant, was sequenced and assembled. It
was inferred that (Kumar et al.
2018) the genome is circular and quadripartite in
structure; it has a length of 154,388 bp and 39.2% GC content and harbored
137 genes including 90 protein-coding genes, 39 tRNAs, and 8 rRNAs. It was
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also added that the genome contained a large single copy of 85,198 bp and a small
single copy of 17,249 bp separated by a pair of inverted repeat regions.
340 S. Pattnaik
18.2 Conclusion
In a nutshell, the medicinal plant cluster can be defined as nature’s best gift for
mankind. But the glaring lacuna is scientific exploration of medicinal plants and the
phytoconstituents produced under stress conditions, in the context of generation of
dataset. Empow ered by the next-generation high throughput, the whole-genome
sequencing conferring both cytoplasmic and plastidial genomes of medicinal plants
is already in the mainstream scientific explorations. The genome sequencing,
followed by assembly, gene ontology annotation, linking to KEGG pathway, and
making a clean and robust dataset for public domain utilization is an important and
acceptable mission. The metabolomics, genomics, and transcriptomics approaches
are the integral part of medicinal plant biology as well as chemistry, in the process of
drug designing and development. Attempts are already lined up by a number of
researchers, bring ing the whole-genome sequence entities, which are openly
accessed as repertoires of functional genes of interest. The catalogued genes are
serving as reference genes (genomes) for de novo sequencing and more so for the
secluded medicinal plants, growing in remote as well as untapped areas. Therefore,
WGS using NGS strategy, of medicinal plants, is considered as a paradigm of
contemporary scientific endeavors.
Acknowledgement This chapter is part of DSc, conferred by Sambalpur University (2018), to the
author.
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345
Targeted Improvement of Medicinal Plant
Under Stress Condition Through
CRISPR-Cas-Mediated Genome Engineering
19
Priyanka Shah, Priya Patel, Manisha Hariwal, Shweta Verma,
Rahul Yadav, and Sanjay Kumar
Abstract
A wide variety of environmental stresses are experienced by plants, which lowers
and limits the yield of agricultural products. Plants experience two different kinds
of environmen tal stress, which are classified as (1) Biotic stress and (2) Abiotic
stress. Major crop plants are lost due to abiotic stress, which includes salinity,
radiation, floods, extreme temperatures, cold, and heavy metals, etc. However,
biotic stressors also include threats from herbivores, bacteria, oomycetes,
nematodes, and other pathogens. To avoid these stresses, scientists develop
genome-editing technology which provides new opportunities for crop improve-
ment and advancement in plant breeding by targeting crop traits. Medicinal plants
are very important as socio-economic purpose. To overcome these stresses,
medicinal plant under stress condition uses CRISPR-Cas technology. It is site-
directed genome editing. Here, we studied the application of CRISPR-Cas to
improve medicinal plant under stress condition.
Keywords
CRISPER/Cas · Medicinal plant · Genetic engineering · Transformation ·
Metabolome engineering
19.1 Introduction
This chapter highlights the importance and future key role of improvement of
medicinal plant under stress condition by using site-directed clustered regularly
interspaced short palindromic repeats (CRISPR)/Cas-mediated genome engineering.
P. Shah · P. Patel · M. Hariwal · S. Verma · R. Yadav · S. Kumar (✉)
Department of Botany, Banaras Hindu University, Varanasi, India
#
The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2023
D. Singh et al. (eds.), Stress-responsive Factors and Molecular Farming
in Medicinal Plants,
https://doi.org/10.1007/978-981-99-4480-4_19
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346 P. Shah et al.
Plants are exposed to various abiotic stresses, viz., ion toxicity, cold, chilling,
heavy metals, radiation, drought, and flood salinity, leading to more than 50% crop
yield loss as a major crop every year (Pandey et al. 2017). Climate change is one of
the most important factors for abiotic stress which may cause a yield loss of more
than 20 percent in future by the year 2050 (Vaughan et al.
2018). To overcome these
challenges, traditional breeding and genetic engineering approaches have been
occupied in the past for generating stress-tolerant crop plants (Chantre Nongpiur
et al.
2016). The traditional genome-editing technology randomly integrates a target
gene into receptor genome; thus it causes poor predictability and problems (Ceasar
et al.
2016). In 2013, the third-generation clustered regularly interspaced short
palindromic repeats (CRISPER)-Cas gene editing system was introduced which
corrected certain defects in first- and second-generation gene editing system based
on the synthetic endonuclease zinc finger nucleases (ZFN) and TALEN (transcrip-
tion activator-like effector nuclease), such as the transfection inefficiency design
complicity and multiplexed mutations (Doudna and Charpentier
2014). The
CRISPR-Cas technology based on the complementary pairing of guide RNA
which sequences with target DNA sequences to identify target sites required only
20 nucleotide sequences to be artificially designed to target specific gene (Lozano-
Juste and Cutler 2014). So CRISPR-Cas system has become the most significant area
for the field of molecular which was used as targeted genetic engineering for many
model crops and plants. In the medicinal plant, this CRISPR-Cas technology has not
been explore as a major level, and the reason behind this is that medicinal plants have
inefficient genetic transformation, regeneration, and genetic complexity.
Medicinal benefit plants have been used for more than thousands of years as a
biological active compo und, pharmaceutical, secondary metabolite, and food sup-
plement. They also have important value in agrochemical, perfume, cosmetics
industry, and anticancer drug.
Some medicinal plants are Salvia miltiorrhiza (Xu et al.
2016), Dendrobium
officinale (Niu et al. 2021), Cannabis sativa (Guo et al. 2018; Yuan et al. 2020), and
Opium poppy (Guo et al. 2018). Scientists are increasingly focused on critical genes
in metabolic pathways and finding novel synthetic methods for increasing the
production of effective compounds (Kiss and Piwowarski
2018
). The application
of CRISPER-Cas system during stress condition modified the gene function study
and metabolic study of medicinal plants which is meaningful and essential,
representing an advanced method for quality improvement and breeding of ideal
germplasm in medicinal plants. At this time introduction of CRISPR-based tools and
their history, mechanism of CRISPR, and its characteristics discusses how they are
used in medicinal plant gene editing, genetic improvement, and various field of
molecular biology.
https://t.me/medicina_free
Соседние файлы в папке Библиотека им академика М.И. Перельмана