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Main conclusion: This study seeks to improve the biomass extractability of Sorghum bicolor by targeting a critical enzyme, 4CL, through metabolic engineering of the lignin biosynthetic pathway at the post-transcriptional level. Sorghum bicolor L., a significant forage crop, offers a potential source of carbohydrate components for biofuel production. The high lignin content in sorghum stems often impedes the extractability of desired carbohydrate components for industrial use. Thus, the present study aimed to develop an improved variety of S. bicolor with reduced lignin through RNA interference of the endogenous 4-coumarate:CoA ligase (4CL) gene involved in the lignin biosynthetic pathway. The S. bicolor gene was isolated, characterized, and used to construct the RNAi-inducing hpRNA gene-silencing construct. Two independent transgenic sorghum lines were produced by introducing an hpRNA-induced gene-silencing cassette of the Sb4CL through Agrobacterium-mediated transformation in the shoot tips of S. bicolor. This was confirmed by PCR amplification of the hygromycin-resistance gene and Southern hybridization. The Sb4CL gene transcript and its enzymatic activity were found to reduce to varying degrees, as shown by northern hybridization and enzyme activity in the independent transgenic samples. Endogenous Sb4CL downregulation in sorghum stem tissue correlates with reduced lignin content to a maximum range of 25%. The transfer of the transgene in the second generation was also analyzed. Decreased lignin content in the transgenic lines was compensated by increased total cell wall carbohydrates such as cellulose (36.56%) and soluble sugars (59.72%) compared to untransformed plants. The study suggests that suppressing the Sb4CL gene effectively develops better sorghum varieties with lower lignin content. This can be useful for industrial purposes, as the enhanced carbohydrate content and favorable alteration of lignin content can lead to economic benefits.

Main conclusion: The evolutionary conservation of type III polyketide synthases (PKS) in Selaginella has been elucidated, and the critical amino acid residues of the anther-specific chalcone synthase-like enzyme (SmASCL) have been identified. Selaginella species are the oldest known vascular plants and a valuable resource for the study of metabolic evolution in land plants. Polyketides, especially flavonoids and sporopollenin precursors, are essential prerequisites for plant land colonization. Although type III polyketide synthases (PKS) are widely studied in seed plants, the related enzymes in Selaginella remain poorly characterized. Here, eight type III PKSs were identified in the Selaginella moellendorffii genome and classified into three clusters. Two PKSs were selected for further research based on their phylogenetic relationships and protein sequence similarity. Functional studies revealed that they were chalcone synthase (SmCHS) and anther-specific CHS-like enzyme (SmASCL). These enzymes are involved in the biosynthesis of flavonoids and sporopollenin, respectively. Their sequence information and enzymatic activity are similar to the orthologs in other plants. Phylogenetic analysis revealed that the ASCL and CHS enzymes were separated into two clades from the Bryophyta. These results suggest that CHS and ASCL emerged in the first land plants and then remained conserved during plant evolution. To study the structural basis of the enzymatic function of SmASCL, a series of mutants were constructed. The number of condensation reactions catalyzed by the P210L/Y211D and I200V/G201T double mutants exceeds that of the wild-type enzyme. Our study provides insight into the characteristics and functions of type III PKSs in S. moellendorffii. It also offers clues for a deeper understanding of the relationship between active sites and the enzymatic function of ASCLs.

Main conclusion: PME12-mutated plants displayed altered stomatal characteristics and susceptibility to ABA-induced closure. Despite changes in PME activity, the mutant exhibited enhanced thermotolerance. These findings suggest a complex interplay between pectin methylesterification, ABA response, and stomatal function, contributing to plant adaptation to heat stress. Pectin, an essential component of plant cell walls, is synthesized in the Golgi apparatus and deposited into the cell wall in a highly methylesterified form. The degree and distribution of methylesterification within homogalacturonan (HGA) domains are crucial in determining its functional properties. Pectin methylesterase (PME) catalyzes the demethylesterification of HGA, which is pivotal for adjusting cell wall properties in response to environmental cues. Our investigation of PME12, a type-I pectin methylesterase in Arabidopsis, reveals its role in abscisic acid (ABA)-mediated stomatal regulation during heat stress, with the pme12 mutant showing increased stomatal density, reduced size, and heightened sensitivity to ABA-induced closure. Additionally, pme12 plants exhibited altered PME activities under heat stress but displayed enhanced thermotolerance. Moreover, our study identified SCRM as a transcriptional regulator positively influencing PME12 expression, linking stomatal development with PME12-mediated pectin methylesterification. These findings suggest that PME12-mediated pectin modification plays a role in coordinating ABA responses and influencing stomatal behavior under heat stress conditions.

Main conclusion: A microRNA with a non-canonical precursor structure harbours an intron in between its miRNA-5p and miRNA-3p relevant for its biogenesis, is conserved across Solanaceae, and targets the mRNA of low phosphate root. Hundreds of miRNAs have been identified in plants and great advances have been accomplished in the understanding of plant miRNA biogenesis, mechanisms and functions. Still, many miRNAs, particularly those with less conventional features, remain to be discovered. Likewise, additional layers of regulation from miRNA generation to action and turnover are still being revealed. The current study describes a microRNA not previously identified given its unusual intron-split stem-loop structure, that has been previously observed only within the monocot-specific miRNA444 family. It shows its conservation across a branch of Solanales including agriculturally relevant Solanaceae family, where its transcripts had already been predicted in several species within sequence databases. The miRNA is absent in Arabidopsis thaliana but present in Solanum lycopersicum, Nicotiana benthamiana, Petunia axillaris, and Ipomoea nil. It proves that at least two different pri-miRNA variants are produced from this miRNA gene, one spliced and the other one retaining the intron. It demonstrates the dual function of its intron in the miRNA biogenesis. On the one hand, its presence in the pri-miRNA positively influences mature miRNA accumulation, but on the other hand, it needs to be removed from the pri-miRNA for efficient mature miRNA production. Finally, it sets low phosphate root as one of its targets, a protein known to be involved in root growth regulation under phosphate starvation in other plant species.

The effects of intense heat during the reproductive phase of two Brassica species-B. napus and C. sativa-could be alleviated by a prior gradual increase exposure and/or PGPR inoculation. Abct. Among extreme weather events caused by climate change, heat waves are one of the most threatening issues for food security. Heat stress is known to be particularly penalizing at the reproductive stage for oleaginous crops, such as oilseed rape and camelina, and is responsible for crop failures as a consequence of yield losses and lower quality of harvest plants parts. In this context, our study aims to analyze two acclimation strategies that rely on the induction of signals prior to an intense heat stress event, i.e., thermopriming (herein, a gradual increase in temperature) and bacteria inoculations (herein, two Plant Growth-Promoting Rhizobacteria (PGPR) were tested). In the two experiments, we assessed the expected beneficial effects of these two acclimation strategies on yield components, seed quality criteria (nutritional and related to dormancy). While thermopriming improved heat stress tolerance in B. napus cv Aviso by maintaining yield, seed nutritional quality and seed dormancy, the effects of the gradual increase prior to the heat stress were even more negative than the later intense heat stress event in C. sativa cv Calena which resulted in cumulated negative effects. The experimentation based on PGPR inoculation highlighted similar trends to thermopriming in B. napus cv Aviso but to a lesser extent. However, in C. sativa cv Calena, very weak effects of PGPR inoculation upon heat stress were observed. Finally, these two acclimation strategies were shown to help alleviate the impacts of intense heat stress but in a species-dependent manner. This study should be deepened by exploring the behaviors of more cultivars of oilseed rape and camelina in the perspective to generalize these results at the species scale.

Main conclusion: The mini-cutting physiological condition is vital for the rooting process. For accurate interpretation, considering all mini-cutting responses in an experiment is necessary to identify significant rooting-biomarkers. The study investigates rooting-biomarkers during vegetative propagation, focusing on Ilex paraguariensis (yerba mate) clones of contrasting mini-cutting rooting performance as a case study (i.e., hard vs. easy). To this end, leaf samples were collected at the time of the rooting experiment and stored in an ultrafreezer. After 120 days, five rooted and five dead cuttings from each clone were selected, and their previously-stored leaves were used for metabolite analysis. Utilizing gas chromatography-mass spectrometry analysis, we identified factors influencing rooting processes, stressing the significance of evaluating both rooted and dead mini-cutting for accurate interpretation. The analysis uncovers 86 compounds, being 47 metabolites identified in the leaves, including vital metabolites such as myo-inositol, sucrose, and 6-kestose, displaying varying concentrations among different clones and their responses. The research underscores the importance of assessing the mini-cutting response to prevent misinterpretations. This was evident in the study, particularly concerning variables like myo-inositol in both clones and chlorogenic acids and 6-kestose in the hard-to-root clone. In summary, the findings highlight the critical role of mini-cutting physiological conditions in the rooting process, providing valuable insights to enhance our understanding of plant propagation techniques.

Main conclusion: AtbZIP69 overexpression in wheat significantly enhanced drought and low nitrogen tolerance by modulating ABA synthesis, antioxidant activity, nitrogen allocation, and transporter gene expression, boosting yield. In this study, we generated wheat plants with improved low nitrogen (LN) and drought tolerance by introducing AtbZIP69, a gene encoding a basic leucine zipper domain transcription factor, into the wheat cultivar Shi 4056. AtbZIP69 localized to the nucleus and activated transcription. A greenhouse study further revealed that, compared to wild type (WT) wheat, AtbZIP69 transgenic wheat exhibited significantly increased drought and LN stress tolerance. Under drought stress, the H₂O₂ concentration in transgenic lines decreased, whereas SOD activity and proline content increased, resulting in remarkably enhanced drought resistance. Furthermore, drought stress boosted the expression of critical abscisic acid (ABA) synthesis enzymes as well as the ABA content of transgenic plants, implying that this gene may improve wheat's drought resistance by promoting ABA production. Additionally, during a two-year field test, the yield and the number of spikes of transgenic wheat were significantly higher than those of WT wheat under LN conditions. Mechanistically, the overexpression of AtbZIP69 altered nitrogen distribution by allocating more nitrogen to grains under LN conditions. In addition, the expression of genes encoding nitrogen transporter proteins was higher in AtbZIP69 transgenic wheat than in WT wheat under LN conditions. These findings suggest that the insertion of AtbZIP69 opens up new opportunities for wheat stress resistance breeding.

Main conclusion: Small RNA sequencing analysis in two chickpea genotypes, JG 62 (Fusarium wilt-susceptible) and WR 315 (Fusarium wilt-resistant), under Fusarium wilt stress led to identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. A miRNA, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. Fusarium wilt (FW) of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris (Foc) is a destructive soil-borne disease that severely reduces the chickpea yield and quality globally. In the present study, we have investigated microRNAs and the microRNA/target gene crosstalk involved in chickpea resistance to FW. The control and stress samples from two genotypes, JG 62 (FW-susceptible) and WR 315 (FW-resistant), collected at 10 days post-inoculation (dpi), were selected for small RNA sequencing. A total of 12 libraries were constructed and sequenced using Illumina HiSeq 2500 platform. The sequencing and in silico analyses revealed the identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 50 miRNAs were physically co-localized with Foc-resistance QTLs present on chromosome 2 (also known as Foc hotspot). A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. Prediction and functional annotation of miRNA targets revealed their role in transcription regulation, disease resistance, defense response, metabolism, etc. Ten miRNAs and their targets were validated using poly(A)-based qRT-PCR in two genotypes grown under lab and field conditions. Many miRNAs and their targets showed genotype-specific expression. The expression profiling also highlighted, both, similar and different expression patterns for the same sets of miRNA and mRNA at different stages of Foc infection. A high correlation in expression patterns of the miRNAs and their targets in lab- and field-grown plant samples was observed. Interestingly, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. The cleavage site in targets was mapped for three miRNAs by analyzing publicly available degradome data for chickpea. The study, for the first time, provides novel insights into microRNA-mediated regulation of resistance and susceptibility mechanisms in chickpea against FW and opens up avenues for the development of the wilt-resistant cultivars in chickpea.

Main conclusion: Rhizobacteria and silicon fertilization synergism suppress leaf and panicle Blast, and mitigates biotic stress in rice plants. Association of bioagents and silicon is synergistic for mitigating leaf and panicle blast and low phosphorus (P) levels in upland rice, under greenhouse conditions. This study aimed to evaluate the potential of the bioagents and silicon interaction on blast disease severity suppression in upland rice plants, under field low P conditions. The experiment was conducted during two growing seasons (E1 and E2), in randomized block design with four replications, and consisted of five treatments, combining a mix of three rhizobacteria, BRM 32114 and BRM62523 (Serratia marcescens), and BRM32110 (Bacillus toyonensis), and three application methods (seed treatment, drenching, spraying). Calcium and magnesium silicate (2 t/ha) was applied over a low soil P, 30 days before sowing. Leaf blast (LBS) and panicle blast (PBS), area under the disease progress curve (AUDPC), activity of enzymes related to oxidative stress, pathogenesis-related (PR), biochemical indicators such as hydrogen peroxide, chlorophyll a and b, carotenoids, and grain yield (GY), were assessed. Bioagents and silicon suppressed LBS by 77.93 and PBS by 62.37%, reduced AUDPC by 77.3 (LBS) and 60.6% (PBS). The yield in E1 was 25% higher than in E2. The treatments statistically differ only in E2, the yield with bioagents and silicon (2435.72 kg ha^-1) was 71.95% higher compared to the absolute control. All enzymatic activities related to oxidative stress and PR proteins were modulated by bioagents and silicon association. The association of rhizobacteria and silicon exhibited a synergistic effect, and represents a bioprotective combination to reduce the effects of different stresses and indirectly reduces the use of chemical inputs.

Main conclusion: Polyploidization (diploidy → polyploidy) was more likely to be positively associated with seed mass than with seed germination. Polyploidy is common in flowering plants, and polyploidization can be associated with the various stages of a plant's life cycle. Our primary aim was to determine the association (positive, none or negative) of polyploidy with seed mass/germination via a literature review. We found that the number of cases of positive, none and negative correlates of polyploidization was 28, 36 and 21, respectively, for seed germination and 25, 5 and 3, respectively, for seed mass. In many plant species, ploidy level differs within and between populations, and it may be positively or negatively associated with germination (57.6% of 85 cases in our review). Ideally, then, to accurately assess intra- and interpopulation variation in seed germination, such studies should include ploidy level. This is the first in-depth review of the association of polyploidy with seed germination.