Young Investigators in Plant Sciences

A special issue of Plants (ISSN 2223-7747).

Deadline for manuscript submissions: closed (10 July 2022) | Viewed by 35638

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Eberhard Karls Universität Tübingen, ZMBP Pflanzenbiochemie - 5th floor, Auf der Morgenstelle 32, D-72076 Tübingen, Germany
Interests: molecular plant-virus interactions; plant defense; plant viruses; geminiviruses; chloroplasts
Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan
Interests: natural products chemistry; drug discovery; antiausterity strategy; pancreatic cancer; biomarker discovery; NMR; structure elucidation; cancer research; chemical biology; metabolomics
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Kansas Lipidomics Research Center, Division of Biology, Kansas State University, Manhattan, KS 66506, USA
Interests: lipid analysis using mass spectrometry; plant lipid metabolism; plant stress responses
Special Issues, Collections and Topics in MDPI journals

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Centro Nacional de Biotecnologia-CSIC, Genetica Molecular de Plantas, Campus Universidad Autonoma, Darwin 3, 28049 Madrid, Spain
Interests: plant viruses; RNA silencing

Special Issue Information

Dear Colleagues,

For the occasion of the 10th anniversary of Plants, it is launching a Special Issue named "Young Investigators in Plants". This Special Issue aims to help young plant scientists to exchange knowledge and increase the visibility of their studies with specialists in any molecular aspect of plant sciences. Through this Special Issue, young researchers are expected to lead the publications derived from their research, either as a first or corresponding author, regardless of whether they are in the early stages of their research careers (undergraduate, graduate, Master, and PhD students) or intermediate stage (postdoctoral researchers or junior group leaders), as long as they meet the requirement of being under 40 years old. Thus, considering the age limitation, this issue will cover a wide variety of areas, aiming to contribute to the overall knowledge of plant sciences regarding several aspects. One novelty compared to previous Special Issues is that, along with the publication, the young author must present a personal profile that includes a photograph, the university from which they have graduated, current affiliation, and interests or scientific background. We aim to reinforce the figure of the young investigators, to give them visibility and to raise interest within the plant science community. With this new initiative, we wish to strengthen the networking of young researchers, paving the way to later stages of their research careers.

Dr. Laura Medina-Puche
Dr. Suresh Awale
Prof. Ruth Welti
Prof. Dr. Juan Antonio García Alvarez
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Plants is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • young investigators
  • plant science
  • plant molecular biology
  • abiotic stress responses
  • plant–pathogen interactions
  • plant development
  • ecology
  • diversity
  • evolution

Published Papers (11 papers)

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13 pages, 2696 KiB  
Article
Stimulation of Germination of Freshly Collected and Cold-Stored Seeds of Ambrosia artemisiifolia L.
by Maja Šćepanović, Laura Košćak, Laura Pismarović and Valentina Šoštarčić
Plants 2022, 11(14), 1888; https://doi.org/10.3390/plants11141888 - 20 Jul 2022
Cited by 1 | Viewed by 1463
Abstract
Herbicides are the most commonly used means of controlling the growth of Ambrosia artemisiifolia L. Their constant use has led to the development of resistant populations. They can be evaluated by studying seed germination and the corresponding grown plants, but A. artemisiifolia exhibits [...] Read more.
Herbicides are the most commonly used means of controlling the growth of Ambrosia artemisiifolia L. Their constant use has led to the development of resistant populations. They can be evaluated by studying seed germination and the corresponding grown plants, but A. artemisiifolia exhibits seed dormancy, preventing germination and delaying research. Here, we developed a simple and rapid method to stimulate germination of freshly collected or stored A. artemisiifolia seeds. The germination of A. artemisiifolia freshly collected/stored seeds was evaluated after storage, stratification, and chemical treatments (ethephon, gibberellic acid (GA3), thiourea, KNO3). Ethephon or ethephon + GA3 improved freshly collected seed germination by 88 and 95%, respectively, and germination of stored seeds by 78 and 80%, respectively. In addition, placing the seeds of A. artemisiifolia in ethephon, GA3, ethephon + GA3, or thiourea solutions caused the freshly collected seeds to germinate faster than stored seeds or nontreated seeds. In contrast, the conditioning of seeds in these solutions favored germination of stored seeds, especially when ethephon + GA3 or GA3 was used. Imbibition of the freshly collected A. artemisiifolia seeds in a mixture of ethephon and GA3 can effectively overcome primary dormancy when rapid experimental results are needed. For seeds requiring prolonged storage, conditioning in ethephon, GA3, or thiourea solutions may be applied to promote germination. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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16 pages, 3849 KiB  
Article
Silicon Enhances Plant Vegetative Growth and Soil Water Retention of Soybean (Glycine max) Plants under Water-Limiting Conditions
by Saroj Kumar Sah, Kambham Raja Reddy and Jiaxu Li
Plants 2022, 11(13), 1687; https://doi.org/10.3390/plants11131687 - 25 Jun 2022
Cited by 4 | Viewed by 2276
Abstract
Silicon has been implicated as a factor affecting the degree of resistance to abiotic stresses in several plant species. However, the role of silicon in soybean (Glycine max) under water-limiting conditions is not yet fully understood. This study was conducted to [...] Read more.
Silicon has been implicated as a factor affecting the degree of resistance to abiotic stresses in several plant species. However, the role of silicon in soybean (Glycine max) under water-limiting conditions is not yet fully understood. This study was conducted to evaluate the effects of silicon application on the vegetative growth of two soybean cultivars (Asgrow 5332 and Progeny 5333) grown under water-limiting conditions. Silicon was provided by adding silicate to the soil. Water-limiting treatments were imposed on plants at two vegetative growth stages for 20 days by irrigating with a reduced amount of water (66% or 33% of the required water). Silicate application enhanced plant height, leaf area, and total dry weight of soybean plants. Significant increases in root volumes were observed in both the silicate-treated cultivars compared to the control plants under water-limiting conditions (33% irrigation). Net photosynthesis and stomatal conductance were decreased, but the quantum efficiency of photosystem II (Fv’/Fm’) did not change under the same irrigation condition, which indicates photosynthesis downregulation through stomatal limitation. Silicate-treated plants in both cultivars had higher water use efficiency as compared to control plants under water-limiting conditions (irrigated with 66% or 33% of required water). Under water-limiting conditions, the soil moisture content was significantly higher in pots containing silicate than in those without added silicate, suggesting that silicon application improves water holding capacity. Taken together, the results from this study indicate that silicon application can improve the vegetative growth of soybeans under low water conditions by increasing the water use efficiency of plants and enhancing the soil’s ability to retain moisture. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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17 pages, 4706 KiB  
Article
Seed-Encapsulation of Desiccation-Tolerant Microorganisms for the Protection of Maize from Drought: Phenotyping Effects of a New Dry Bioformulation
by Inês Rebelo Romão, Ana Sofia Rodrigues dos Santos, Leonardo Velasco, Elsa Martínez-Ferri, Juan Ignacio Vilchez and Maximino Manzanera
Plants 2022, 11(8), 1024; https://doi.org/10.3390/plants11081024 - 09 Apr 2022
Cited by 1 | Viewed by 2216
Abstract
Droughts and high temperatures deeply affect crop production. The use of desiccation-tolerant (or xerotolerant) microorganisms able to protect plants from droughts represents a promising alternative. These xerotolerant microorganisms have previously been used to modulate plant responses and improve their tolerance to drought. In [...] Read more.
Droughts and high temperatures deeply affect crop production. The use of desiccation-tolerant (or xerotolerant) microorganisms able to protect plants from droughts represents a promising alternative. These xerotolerant microorganisms have previously been used to modulate plant responses and improve their tolerance to drought. In addition, these microorganisms could be stored and used in dry formats, which would improve their viability and resilience at a much lower cost than current market alternatives. In the present study we analyze the possibility of using strains of xerotolerant Actinobacteria in encapsulated format on seeds. Under this formulation, we carried out greenhouse with farming soil with maize plants. Under greenhouse conditions, the plants showed greater resistance to drought, as well as increased growth and production yield, but not as well in field trials. This alternative could represent a useful tool to improve water efficiency in crops for drought-affected areas or affected by water scarcity. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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10 pages, 840 KiB  
Article
DNA Barcoding Study of Representative Thymus Species in Bulgaria
by Ina Aneva, Petar Zhelev, Georgi Bonchev, Irina Boycheva, Stiliana Simeonova and Denitsa Kancheva
Plants 2022, 11(3), 270; https://doi.org/10.3390/plants11030270 - 20 Jan 2022
Cited by 7 | Viewed by 2013
Abstract
We present a study on the taxonomy of eleven Thymus species, belonging to two sections and occurring naturally in Bulgaria. Four DNA barcoding markers—matK, rbcL, trnH-psbA and ITS—were applied to discriminate the species and to reveal their phylogenetic relationships. The results showed that [...] Read more.
We present a study on the taxonomy of eleven Thymus species, belonging to two sections and occurring naturally in Bulgaria. Four DNA barcoding markers—matK, rbcL, trnH-psbA and ITS—were applied to discriminate the species and to reveal their phylogenetic relationships. The results showed that rbcL has the lowest discriminating power regarding the studied species, while the other markers yielded results fitting better to the existing taxonomic schemes based on morphological traits. However, even in the case of better performing markers, the results were not straightforward—morphologically distinct species belonging to different sections were grouped together, and closely related species appeared genetically distinct. The results are typical for taxonomically complex groups, such as the genus Thymus, characterized in Bulgaria with great diversity, high percentage of endemism and still requiring a full and comprehensive taxonomic study. The results are discussed in the light of unresolved taxonomic problems and application of DNA barcoding methods. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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12 pages, 3498 KiB  
Article
Different LED Light Wavelengths and Photosynthetic Photon Flux Density Effect on Colletotrichum acutatum Growth
by Neringa Rasiukevičiūtė, Aušra Brazaitytė, Viktorija Vaštakaitė-Kairienė and Alma Valiuškaitė
Plants 2022, 11(1), 143; https://doi.org/10.3390/plants11010143 - 05 Jan 2022
Viewed by 3043
Abstract
The study aimed to evaluate the effect of different photon flux density (PFD) and light-emitting diodes (LED) wavelengths on strawberry Colletotrichum acutatum growth characteristics. The C. acutatum growth characteristics under the blue 450 nm (B), green 530 nm (G), red 660 nm (R), [...] Read more.
The study aimed to evaluate the effect of different photon flux density (PFD) and light-emitting diodes (LED) wavelengths on strawberry Colletotrichum acutatum growth characteristics. The C. acutatum growth characteristics under the blue 450 nm (B), green 530 nm (G), red 660 nm (R), far-red 735 nm (FR), and white 5700 K (W) LEDs at PFD 50, 100 and 200 μmol m2 s−1 were evaluated. The effect on C. acutatum mycelial growth evaluated by daily measuring until five days after inoculation (DAI). The presence of conidia and size (width and length) evaluated after 5 DAI. The results showed that the highest inhibition of fungus growth was achieved after 1 DAI under B and G at 50 μmol m−2 s−1 PFD. Additionally, after 1–4 DAI under B at 200 μmol m−2 s−1 PFD. The lowest conidia width was under FR at 50 μmol m−2 s−1 PFD and length under FR at 100 μmol m−2 s−1 PFD. Various LED light wavelengths influenced differences in C. acutatum colonies color. In conclusion, different photosynthetic photon flux densities and wavelengths influence C. acutatum growth characteristics. The changes in C. acutatum morphological and phenotypical characteristics could be related to its ability to spread and infect plant tissues. This study’s findings could potentially help to manage C. acutatum by LEDs in controlled environment conditions. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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22 pages, 4661 KiB  
Article
Differences in the Abundance of Auxin Homeostasis Proteins Suggest Their Central Roles for In Vitro Tissue Differentiation in Coffea arabica
by Ana O. Quintana-Escobar, Hugo A. Méndez-Hernández, Rosa M. Galaz-Ávalos, José M. Elizalde-Contreras, Francisco A. Reyes-Soria, Victor Aguilar-Hernández, Eliel Ruíz-May and Víctor M. Loyola-Vargas
Plants 2021, 10(12), 2607; https://doi.org/10.3390/plants10122607 - 27 Nov 2021
Cited by 3 | Viewed by 3288
Abstract
Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem [...] Read more.
Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem mass tag (TMT) and the synchronous precursor selection (SPS)-based MS3 technology on the Orbitrap Fusion™ Tribrid mass spectrometer™ in three types of biological materials corresponding to C. arabica: plantlet leaves, calli, and suspension cultures. Proteins included in the β-oxidation of indole butyric acid and in the signaling, transport, and conjugation of indole-3-acetic acid were identified, such as the indole butyric response (IBR), the auxin binding protein (ABP), the ATP-binding cassette transporters (ABC), the Gretchen-Hagen 3 proteins (GH3), and the indole-3-acetic-leucine-resistant proteins (ILR). A more significant accumulation of proteins involved in auxin homeostasis was found in the suspension cultures vs. the plantlet, followed by callus vs. plantlet and suspension culture vs. callus, suggesting important roles of these proteins in the cell differentiation process. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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24 pages, 7152 KiB  
Article
Sunscreen Effect Exerted by Secondary Carotenoids and Mycosporine-like Amino Acids in the Aeroterrestrial Chlorophyte Coelastrella rubescens under High Light and UV-A Irradiation
by Anna Zaytseva, Konstantin Chekanov, Petr Zaytsev, Daria Bakhareva, Olga Gorelova, Dmitry Kochkin and Elena Lobakova
Plants 2021, 10(12), 2601; https://doi.org/10.3390/plants10122601 - 26 Nov 2021
Cited by 18 | Viewed by 3109
Abstract
The microalga Coelastrella rubescens dwells in habitats with excessive solar irradiation; consequently, it must accumulate diverse compounds to protect itself. We characterized the array of photoprotective compounds in C. rubescens. Toward this goal, we exposed the cells to high fluxes of visible [...] Read more.
The microalga Coelastrella rubescens dwells in habitats with excessive solar irradiation; consequently, it must accumulate diverse compounds to protect itself. We characterized the array of photoprotective compounds in C. rubescens. Toward this goal, we exposed the cells to high fluxes of visible light and UV-A and analyzed the ability of hydrophilic and hydrophobic extracts from the cells to absorb radiation. Potential light-screening compounds were profiled by thin layer chromatography and UPLC-MS. Coelastrella accumulated diverse carotenoids that absorbed visible light in the blue–green part of the spectrum and mycosporine-like amino acids (MAA) that absorbed the UV-A. It is the first report on the occurrence of MAA in Coelastrella. Two new MAA, named coelastrin A and coelastrin B, were identified. Transmission electron microscopy revealed the development of hydrophobic subcompartments under the high light and UV-A exposition. We also evaluate and discuss sporopollenin-like compounds in the cell wall and autophagy-like processes as the possible reason for the decrease in sunlight absorption by cells, in addition to inducible sunscreen accumulation. The results suggested that C. rubescens NAMSU R1 accumulates a broad range of valuable photoprotective compounds in response to UV-A and visible light irradiation, which indicates this strain as a potential producer for biotechnology. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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15 pages, 1655 KiB  
Article
Evaluation of Frost Damage and Pod Set in Faba Bean (Vicia faba L.) under Field Conditions
by Najeeb H. Alharbi, Salem S. Alghamdi, Hussein M. Migdadi, Ehab H. El-Harty and Kedar N. Adhikari
Plants 2021, 10(9), 1925; https://doi.org/10.3390/plants10091925 - 15 Sep 2021
Cited by 6 | Viewed by 2102
Abstract
Frost is one factor that causes extensive yield losses globally. A study was conducted to evaluate frost damage under field conditions and assess the genetic variation of flowers converting into pods. Diverse faba bean genotypes were evaluated under four growing seasons in a [...] Read more.
Frost is one factor that causes extensive yield losses globally. A study was conducted to evaluate frost damage under field conditions and assess the genetic variation of flowers converting into pods. Diverse faba bean genotypes were evaluated under four growing seasons in a randomized complete block design: three at the University of Sydney, Narrabri, Australia (2014–2016) with three sowing dates, and one at the Agricultural Research Station, Dirab, Riyadh, Saudi Arabia (2016/2017) in one sowing. Visual methods were used to estimate frost damage and record the development of pods. Radiation frost in 2014 (Narrabri) damaged lower pods, while advection frost in 2016/2017 (Dirab) damaged upper pods. The radiation frost formed immediately above the ground; therefore, flowers and pods of taller plants minimized the damage because of their long distance from the ground. The earliest (mid-April) and middle sowing (7 May) suffered more by frost, while a delay in sowing (last week in May) led to frost escape or minor damage. The genotypes IX474/4-3 and 11NF010a-2 showed low sensitivity to frost at the vegetative and reproductive stages. Flowers developed at the beginning of flowering had a faster and higher pod formation rate (41–43%) than those formed later and contributed more to yields. Therefore, a severe frost at the beginning of flowering can cause a significant yield loss as these flowers are the most productive. The frost-tolerant genotypes, and faster and higher pod forming rates, identified in this study can be exploited to breed better varieties in the future. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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17 pages, 2356 KiB  
Article
Protein, Amino Acid, Oil, Fatty Acid, Sugar, Anthocyanin, Isoflavone, Lutein, and Antioxidant Variations in Colored Seed-Coated Soybeans
by Sanjeev Kumar Dhungana, Jeong-Hyun Seo, Beom-Kyu Kang, Ji-Hee Park, Jun-Hoi Kim, Jung-Sook Sung, In-Youl Baek, Sang-Ouk Shin and Chan-Sik Jung
Plants 2021, 10(9), 1765; https://doi.org/10.3390/plants10091765 - 25 Aug 2021
Cited by 9 | Viewed by 2629
Abstract
Different physiological and genetic studies show that the variations in the accumulation of pigment-stimulating metabolites result in color differences in soybean seed coats. The objective of this study was to analyze the nutrient contents and antioxidant potential in black, brown, and green seed-coated [...] Read more.
Different physiological and genetic studies show that the variations in the accumulation of pigment-stimulating metabolites result in color differences in soybean seed coats. The objective of this study was to analyze the nutrient contents and antioxidant potential in black, brown, and green seed-coated soybeans. Significant variations in protein (38.9–43.3%), oil (13.9–20.4%), total sugar (63.5–97.0 mg/g seed), total anthocyanin (3826.0–21,856.0 μg/g seed coat), total isoflavone (709.5–3394.3 μg/g seed), lutein (1.9–14.8 μg/g), total polyphenol (123.0–385.8 mg gallic acid/100 g seed), total flavonoid (22.1–208.5 mg catechin/100 g seed), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS; 275.0–818.8 mg Trolox/100 g seed), and 2,2-diphenyl-1-picrylhydrazyl (DPPH; 96.3–579.7 mg Trolox/100 g seed) were found among the soybean genotypes. Ilpumgeomjeong2 contained the lowest protein but the highest oil and total sugar. The lowest oil-containing Wonheug had the highest protein content. Socheong2 was rich in all four variables of antioxidants. Anthocyanins were detected only in black soybeans but not in brown and green soybeans. The variation in isoflavone content was up to 5-fold among the soybean genotypes. This study could be a valuable resource for the selection and improvement of soybean because an understanding of the nutrient content and antioxidant potentials is useful to develop effective strategies for improving the economic traits; for example, the major emphasis of soybean breeding for fatty acids is to enhance the oleic and linoleic acid contents and to decrease linolenic acid content. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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14 pages, 1269 KiB  
Article
Differential Responses of Antioxidative System during the Interaction of Soursop Fruits (Annona muricata L.) and Nectria haematococca at Postharvest Storage
by Alejandro Rubio-Melgarejo, Rosendo Balois-Morales, Verónica Alhelí Ochoa-Jiménez, Paloma Patricia Casas-Junco, José Orlando Jiménez-Zurita, Pedro Ulises Bautista-Rosales and Guillermo Berumen-Varela
Plants 2021, 10(7), 1432; https://doi.org/10.3390/plants10071432 - 14 Jul 2021
Cited by 8 | Viewed by 3177
Abstract
Soursop fruit (Annona muricata L.) production is diminished by the attack of pathogens such as Nectria haematococca. However, the fruit–pathogen interaction at the biochemical and molecular levels is still unknown. The objective of this study was to analyze the response of [...] Read more.
Soursop fruit (Annona muricata L.) production is diminished by the attack of pathogens such as Nectria haematococca. However, the fruit–pathogen interaction at the biochemical and molecular levels is still unknown. The objective of this study was to analyze the response of the soursop fruit to the presence of N. haematococca during postharvest storage. Soursop fruits were inoculated with the pathogen and total phenolic compounds, antioxidant capacity by Ferric reducing/antioxidant power (FRAP), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), as well as enzymatic activity and transcript levels of polyphenol oxidase (PPO) and superoxide dismutase (SOD), were evaluated at 1, 3, and 5 days of storage. The noninoculated fruits were the controls of the experiment. The highest total phenol content was recorded on day one in the inoculated fruits. FRAP, ABTS, and DPPH activity presented the highest values on day three in the control fruits. Inoculated fruits recorded the highest PPO activity on day five and a five-fold induction in the PPO transcript on day three. SOD activity showed a decrease during the days of storage and 10-fold induction of SOD transcript on day three in the inoculated fruits. Principal component analysis showed that total phenols were the variable that contributed the most to the observed variations. Furthermore, a positive correlation between total phenols and SOD activity, PPO expression, and SOD expression, as well as between DPPH and FRAP, was recorded. The results showed a differential response in antioxidant capacity, enzymatic activity, and gene expression during the interaction of soursop fruits–N. haematococca at postharvest storage. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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17 pages, 3087 KiB  
Study Protocol
Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean
by Won-Nyeong Kim, Hye-Jeong Kim, Young-Soo Chung and Hyun-Uk Kim
Plants 2021, 10(11), 2542; https://doi.org/10.3390/plants10112542 - 22 Nov 2021
Cited by 6 | Viewed by 3967
Abstract
CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 [...] Read more.
CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants. Full article
(This article belongs to the Special Issue Young Investigators in Plant Sciences)
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