Exploiting Beneficial Pseudomonas spp. for Cannabis Production

Among the oldest domesticated crops, cannabis plants (Cannabis sativa L., marijuana and hemp) have been used to produce food, fiber, and drugs for thousands of years. With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high-value market is emerging for the supply of marijuana and hemp products. This creates unprecedented challenges to achieve better yields and environmental sustainability, while lowering production costs. In this review, we discuss the opportunities and challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop inoculants to improve productivity. The prevalence and diversity of naturally occurring Pseudomonas strains within the cannabis microbiome is overviewed, followed by their potential mechanisms involved in plant growth promotion and tolerance to abiotic and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and marijuana crops in various production systems. Finally, factors likely to influence inoculant efficacy are provided, along with strategies to identify promising strains, overcome commercialization bottlenecks, and design adapted formulations. This work aims at supporting the development of the cannabis industry in a sustainable way, by exploiting the many beneficial attributes of Pseudomonas spp.

​Standardization of Grafting in Bittergourd (Momordica charantia L.)

Background: Commercial cultivation of bitter gourd is affected by biotic stresses like mosaic disease, fusarium wilt and root-knot nematode as well as abiotic stress like drought. Grafting with resistant rootstocks can be a tool to control these problems. In vegetable production, grafting is exploited commercially in many parts of the world. The cultivated area of grafted solanaceae and cucubitaceae plants has increased tremendously in recent years because of the advantages of grafted plants. Commercial use of vegetable grafting is a relatively recent innovation in India and scientific information on grafting in bittergourd is meager. In this context, identification of suitable rootstocks and standardization of grafting techniques that do not have adverse effect on yield and fruit quality not only lay foundation for further evaluation on tolerance to different biotic and abiotic stresses but also enhance the area and production of bitter gourd especially in sustainable production systems. Methods: Grafting in bitter gourd was carried out with three grafting methods such as hole insertion grafting, one cotyledon grafting and cleft grafting using growth regulators viz., alar and CCC to control height of rootstocks in order to identify suitable method, growth regulator and its concentration. The grafting experiment was done independently to four cucurbitaceous rootstocks viz., sponge gourd, pumpkin, bottle gourd and bitter gourd using bittergourd var. Preethi as scion. Height and diameter of the rootstocks before grafting were recorded and then the growth regulators alar and CCC each at 10 mgL-1 and 50 mgL-1 along with distilled water as control were sprayed on rootstocks in order to prevent the lodging of the root stocks and then grafted using different methods. Days taken for graft union and percentage success were also evaluated after grafting. Result: Our study of grafting bitter gourd scion into four cucurbitaceous rootstocks utilizing three methods and two growth regulators at two different concentration along with control exhibited significant difference in graft success among the methods as well as concentration of growth regulator in all four experiments. This work can be further utilized for imparting resistance against abiotic and biotic stresses in bitter gourd by selecting suitable rootstocks.

Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

Biostimulants in Viticulture: A Sustainable Approach against Biotic and Abiotic Stresses

Climate change and disproportionate anthropogenic interventions, such as the excess of phytopharmaceutical products and continuous soil tillage, are jeopardizing viticulture by subjecting plants to continuous abiotic stress. One of the main physiological repercussions of abiotic stress is represented by the unbalanced redox homeostasis due to the overproduction of reactive oxygen species (ROS), ultimately leading to a state of oxidative stress (detrimental to grape quality). To these are added the direct and indirect damages caused by pathogens (biotic stresses). In light of this scenario, it is inevitable that sustainable techniques and sensitivity approaches for environmental and human health have to be applied in viticulture. Sustainable viticulture can only be made with the aid of sustainable products. Biostimulant (PB) applications (including resistance inducers or elicitors) in the vineyard have become interesting maneuvers for counteracting vine diseases and improving grape quality. These also represent a partial alternative to soil fertilization by improving nutrient absorption and avoiding its leaching into the groundwater. Their role as elicitors has important repercussions in the stimulation of the phenylpropanoid pathway by triggering the activation of several enzymes, such as polyphenol oxidase, lipoxygenase, phenylalanine ammonia-lyase, and peroxidase (with the accumulation of phenolic compounds). The present review paper summarizes the PBs’ implications in viticulture, gathering historical, functional, and applicative information. This work aims to highlight the innumerable beneficial effects on vines brought by these products. It also serves to spur the scientific community to a greater contribution in investigating the response mechanisms of the plant to positive inductions.

The Physiological Impact of GFLV Virus Infection on Grapevine Water Status: First Observations

In a vineyard, grapevines are simultaneously exposed to combinations of several abiotic (drought, extreme temperatures, salinity) and biotic stresses (phytoplasmas, viruses, bacteria). With climate change, the incidences of drought in vine growing regions are increased and the host range of pathogens with increased chances of virulent strain development has expanded. Therefore, we studied the impact of the combination of abiotic (drought) and biotic (Grapevine fanleaf virus (GFLV) infection) stress on physiological and molecular responses on the grapevine of cv. Schioppettino by studying the influence of drought and GFLV infection on plant water status of grapevines, on grapevine xylem vessel occlusion, and on expression patterns of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1), 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), WRKY encoding transcription factor (WRKY54) and RD22-like protein (RD22) genes in grapevines. A complex response of grapevine to the combination of drought and GFLV infection was shown, including priming in the case of grapevine water status, net effect in the case of area of occluded vessels in xylem, and different types of interaction of both stresses in the case of expression of four abscisic acid-related genes. Our results showed that mild (but not severe) water stress can be better sustained by GFLV infection rather than by healthy vines. GFLV proved to improve the resilience of the plants to water stress, which is an important outcome to cope with the challenges of global warming.

Characterization of histone deacetylases and their roles in response to abiotic and PAMPs stresses in Sorghum bicolor

Background Histone deacetylases (HDACs) play an important role in the regulation of gene expression, which is indispensable in plant growth, development, and responses to environmental stresses. In Arabidopsis and rice, the molecular functions of HDACs have been well-described. However, systematic analysis of the HDAC gene family and gene expression in response to biotic and abiotic stresses has not been reported for sorghum. Results We conducted a systematic analysis of the sorghum HDAC gene family and identified 19 SbHDACs mainly distributed on eight chromosomes. Phylogenetic tree analysis of SbHDACs showed that the gene family was divided into three subfamilies: RPD3/HDA1, SIR2, and HD2. Tissue-specific expression results showed that SbHDACs displayed different expression patterns in different tissues, indicating that these genes may perform different functions in growth and development. The expression pattern of SbHDACs under different stresses (high and low temperature, drought, osmotic and salt) and pathogen-associated molecular model (PAMPs) elf18, chitin, and flg22) indicated that SbHDAC genes may participate in adversity responses and biological stress defenses. Overexpression of SbHDA1, SbHDA3, SbHDT2 and SbSRT2 in Escherichia coli promoted the growth of recombinant cells under abiotic stress. Interestingly, we also showed that the sorghum acetylation level was enhanced when plants were under cold, heat, drought, osmotic and salt stresses. The findings will help us to understand the HDAC gene family in sorghum, and illuminate the molecular mechanism of the responses to abiotic and biotic stresses. Conclusion We have identified and classified 19 HDAC genes in sorghum. Our data provides insights into the evolution of the HDAC gene family and further support the hypothesis that these genes are important for the plant responses to abiotic and biotic stresses.

SlWRKY45 Interacts with Jasmonate-ZIM Domain Proteins to Negatively Regulate Defense Against Root Knot Nematode Meloidogyne incognita in Tomato

Root knot nematode (RKN), a kind of plant parasitic nematodes, leads to large reduction of crop yield, and seriously damages the agricultural production. The phytohormone jasmonates (JAs) act as important signals to regulate resistance against multiple abiotic and biotic stresses. However, little is known about the mechanism of JA-mediated defense responses against RKN in tomato. In this study, we found that the WRKY transcription factor SlWRKY45 interacts with most of the Jasmonate-ZIM domain proteins (JAZs) in yeast and plant. Overexpression of SlWRKY45 decreased plant resistance to RKN Meloidogyne incognita with increased gall index. We further generated slwrky45 mutants using the CRISPR/Cas9 technology, and discovered that the gall index and the number of nematodes and females in slwrky45 mutants are significantly reduced compared with wild type, as inoculated with RKN Meloidogyne incognita. Moreover, the contents of jasmonic acid and JA-isoleucine (JA-Ile) were highly increased in slwrky45 mutants with RKN Meloidogyne incognita infection compared with wild type. Furthermore, EMSA, and Dual-LUC assays demonstrated that SlWRKY45 directly binds and represses jasmonate biosynthesis gene ALLENE OXIDE CYCLASE ( AOC). Overall, our findings reveled that JAZ-interaction protein SlWRKY45 negatively controls plant defense against RKN Meloidogyne incognita by the regulation of JA biosynthesis in tomato.

Genome-Wide Prediction, Functional Divergence, and Characterization of Stress-Responsive BZR Transcription Factors in B. napus

BRASSINAZOLE RESISTANT (BZR) are transcriptional factors that bind to the DNA of targeted genes to regulate several plant growth and physiological processes in response to abiotic and biotic stresses. However, information on such genes in Brassica napus is minimal. Furthermore, the new reference Brassica napus genome offers an excellent opportunity to systematically characterize this gene family in B. napus. In our study, 21 BnaBZR genes were distributed across 19 chromosomes of B. napus and clustered into four subgroups based on Arabidopsis thaliana orthologs. Functional divergence analysis among these groups evident the shifting of evolutionary rate after the duplication events. In terms of structural analysis, the BnaBZR genes within each subgroup are highly conserved but are distinctive within groups. Organ-specific expression analyses of BnaBZR genes using RNA-seq data and quantitative real-time polymerase chain reaction (qRT-PCR) revealed complex expression patterns in plant tissues during stress conditions. In which genes belonging to subgroups III and IV were identified to play central roles in plant tolerance to salt, drought, and Sclerotinia sclerotiorum stress. The insights from this study enrich our understanding of the B. napus BZR gene family and lay a foundation for future research in improving rape seed environmental adaptability.

The Evolution and Functional Roles of miR408 and Its Targets in Plants

MicroRNA408 (miR408) is an ancient and highly conserved miRNA, which is involved in the regulation of plant growth, development and stress response. However, previous research results on the evolution and functional roles of miR408 and its targets are relatively scattered, and there is a lack of a systematic comparison and comprehensive summary of the detailed evolutionary pathways and regulatory mechanisms of miR408 and its targets in plants. Here, we analyzed the evolutionary pathway of miR408 in plants, and summarized the functions of miR408 and its targets in regulating plant growth and development and plant responses to various abiotic and biotic stresses. The evolutionary analysis shows that miR408 is an ancient and highly conserved microRNA, which is widely distributed in different plants. miR408 regulates the growth and development of different plants by down-regulating its targets, encoding blue copper (Cu) proteins, and by transporting Cu to plastocyanin (PC), which affects photosynthesis and ultimately promotes grain yield. In addition, miR408 improves tolerance to stress by down-regulating target genes and enhancing cellular antioxidants, thereby increasing the antioxidant capacity of plants. This review expands and promotes an in-depth understanding of the evolutionary and regulatory roles of miR408 and its targets in plants.