Stages Of Plant Development Research Articles

Phytotoxicity Assessment of Agro-Industrial Waste and Its Biochar: Germination Bioassay in Four Horticultural Species

This study investigated the phytotoxicity of agro-industrial wastes (almond, walnut, pistachio and peanut shells, asparagus spears, and brewer’s spent grain) and their biochar through germination bioassays in several horticultural species: green pea, lettuce, radish, and arugula. Biowaste was pyrolyzed under controlled conditions to produce biochar, and both biowaste and biochar were characterized. Germination bioassay was conducted using seeds exposed to different dilutions of aqueous extract of biowaste and their biochar (0, 50, and 100%). Germination percentage, seed vigor, germination index, and root and aerial lengths were evaluated. The results showed that the phytotoxicity of the biowaste was significantly different to that of its biochar. The biochar obtained demonstrated changing effects on germination and seedling growth. In particular, biochar extracts from spent brewers grains, walnut shells, and pistachio shells showed 5–14% increases in seed vigor and root and aerial length. Furthermore, the response of different species to both agro-industrial waste and biochar revealed species-specific sensitivity. Seeds of lettuce and arugula species were more sensitive to aqueous extracts than radish and green peas. This knowledge not only elucidates the behavior of agro-industrial waste-based biochar in the early stage of plant development but also provides valuable insights regarding phytotoxicity, seed sensitivity, and the variables involved in germination.

More than flowering: CONSTANS plays multifaceted roles in plant development and stress responses.

Plants have evolved a remarkable ability to sense and respond to changes in photoperiod, allowing adjustments to their growth and development based on seasonal and environmental cues. The floral transition is a pivotal stage in plant growth and development, signifying a shift from vegetative to reproductive growth. CONSTANS (CO), a central photoperiodic response factor conserved in various plants, mediates day-length signals to control the floral transition, although its mechanisms of action vary among plants with different day-length requirements. In addition, recent studies have uncovered roles for CO in organ development and stress responses. These pleiotropic roles in model plants and crops make CO a potentially fruitful target for molecular breeding aimed at modifying crop agronomic traits. This review systematically traces research on CO, from its discovery and functional studies to the exploration of its regulatory mechanisms and newly discovered functions, providing important insight into the roles of CO and laying a foundation for future research.

Bell pepper endornavirus alters green peach aphid (Hemiptera: Aphididae) host choice and population dynamics.

Bell pepper endornavirus (BPEV) Alphaendornavirus capsici (Endornaviridae) is an RNA virus that infects many pepper (Capsicum annuum) horticultural types and is seed transmitted. BPEV does not cause apparent symptoms and is found at every plant developmental stage. During the domestication of bell pepper, plant breeders, unaware of the existence of endornaviruses in the germplasm, selected endornavirus-infected genotypes. This could be an indication that the presence of endornaviruses in this crop is beneficial. Among the possible beneficial effects that endornaviruses may provide to their host could include tolerance or resistance to biotic and abiotic agents and, therefore, may have evolved a symbiotic relationship with their hosts. With this in mind, we set out to determine host preference, host suitability, and population dynamics of green peach aphid Myzus persicae (Sulzer) on BPEV-infected and virus-free bell pepper near-isogenic lines. During choice bioassay experiments, we observed that a higher proportion of M. persicae adults settled on BPEV noninfected leaves as compared to BPEV-infected leaves. Life table analysis revealed that M. persicae performed less well on BPEV-infected leaf tissues, with reductions in longevity, progeny, and intrinsic rate of increase. These results indicate BPEV is beneficial to its host, protecting against an important generalist pest.

Association mapping unravels the genetic basis for drought related traits in different developmental stages of barley

Drought stress significantly reduces crop yields at all stages of plant development. Barley, known for its abiotic-stress adaptation among cereals was used to examine the genetic basis of drought tolerance. A population of 164 spring barley lines was subjected to polyethylene glycol (PEG) induced drought stress during germination and seedling development. Six traits were measured, including germination percentage and rate, seedling length and weight, and root-to-shoot ratios. Seedling area, volume, and root and shoot diameter was acquired with a flatbed scanner. This population was also subjected to short-term drought during the heading stage in the greenhouse. Root and shoot weight and grain yield data were collected from well watered and droughted plants. Significant variation within traits were observed and several of them exhibited strong correlations with each other. In this population, two genotypes had 100% germination under PEG-induced drought and drought tolerance throughout the heading stage of plant development. A genome-wide association scan (GWAS) revealed 64 significant marker-trait associations across all seven barley chromosomes. Candidate genes related to abiotic stress and germination were identified within a 0.5Mbp interval around these SNPs. In silico analysis indicated a high frequency of differential expression of the candidate genes in response to stress. This study enabled identification of barley lines useful for drought tolerance breeding and pinpointed candidate genes for enhancing drought resiliency in barley.

Gene Expression Analysis for Drought Tolerance in Early Stage of Potato Plant Development

Drought has increasingly affected the yield of Solanum tuberosum L. (potato) every year over the last decade, posing serious economic problems for the global agricultural industry. Therefore, it is important to research drought tolerance in plants and obtain more robust varieties of crops. The aim of the present work was to study the expression of drought-upregulated genes in drought-tolerant and drought-sensitive varieties of potato. Bioreactors were used to identify whether each variety was drought-tolerant or drought-sensitive; then, expression analysis was performed according to the morphological characteristics of the plantlets in two different media: Murashige and Skoog (MS) medium and MS medium with 20% PEG-6000 to simulate osmotic stress. Based on the quantitative parameters of six initial varieties, two varieties were selected (Gala and Aksor) for further gene expression analysis. The expression of genes commonly upregulated in drought (ER24, TAS14, DREB147315, PP2C, 102605413 and NF-YC4) was higher in the drought-tolerant variety than in the sensitive one. Therefore, the expression of these genes can be used to determine the drought tolerance of a potato variety in vitro in the early plant development stage. Moreover, comparative analysis showed that some of the targeted genes used to identify drought tolerance in this study are conserved across different plant species.

Effects of green seaweed (<i>Ulva onhoi</i>) on the reproductive development of tomato (<i>Solanum lycopersicum)</i> plants

Algae-derived products have great potential as crop biostimulants due to their multiple beneficial effects at different stages of plant development. Green seaweeds of the genus <i>Ulva</i> are well suited for this purpose because they are widely distributed and grow rapidly in a wide range of conditions. In this study, a greenhouse experiment was conducted to evaluate the effects of dry seaweed powder (DSP) and liquid seaweed extract (LSE) of <i>Ulva ohnoi</i> on the reproductive development of tomato plants. The experiment included three treatments: 1) plants treated with 5 g of DSP, 2) plants treated with 250 mL of LSE, and (3) control plants (without algae). The reproductive parameters, chlorophyll content, and mineral composition were measured during the flowering and early fruiting periods. The application of DSP was the most effective treatment in promoting early flowering and significantly increased the number of buds (103%), flower clusters (55%), flowers (61%), and fruits (45%) per plant. Furthermore, the DSP-treated plants exhibited an enhancement in the levels of chlorophyll and nutrients in the plants and fruits. The results of the current work show that the application of <i>U. ohnoi</i> in its natural form (dry powder) stimulates the reproductive development of crop tomato. This represents a sustainable and natural alternative to synthetic inputs that growers can incorporate into horticultural production to improve yield attributes.

Opportunities and limits of image-based plant stress phenotyping: detecting plant salt stress status using machine learning techniques

Abstract Plant stress phenotyping is a useful tool to facilitate targeted interventions and optimize management practices of plants growing in stressful conditions. However, various technical challenges still need to be overcome, though, and there is a clear need for investigating functional traits that can be used as a proxy for stress prediction, especially for abiotic stresses. This experimental work leveraged machine learning classification models to detect salt stress in two populations of a non-model species, combining image-based approaches (i.e. both manual and automated) and minimal morpho-physiological/biochemical analyses. A small set of specific features, combining malondialdehyde content with other non-destructive image-derived traits, such as Chroma Difference and Chroma Ratio indices, was able to distinguish non-stressed from stressed plants (2-class model; precision: 0.91) as well as stress intensity (3-class model; precision: 0.84). This flexible approach can be adapted to different plant developmental stages and leaf shape/morphology. In the future, the robustness and reliability of the models should be tested in other species and other abiotic stresses, such as drought.

Hydrogen peroxide participates in leaf senescence by inhibiting CHLI1 activity.

Hydrogen peroxide promoted leaf senescence by sulfenylating the magnesium chelating protease I subunit (CHLI1) in the chlorophyll synthesis pathway, and inhibited its activity to reduce chlorophyll synthesis. Leaf senescence is the final and crucial stage of plant growth and development, during which chlorophyll experiences varying degrees of destruction. It is well-known that the higher ROS accumulation is a key factor for leaf senescence, but whether and how ROS regulates chlorophyll synthesis in the process are unknown. Here, we report that H2O2 inhibits chlorophyll synthesis during leaf senescence via the I subunit of magnesium-chelatase (CHLI1). During leaf senescence, the decrease of chlorophyll content is accompanied by the increase of H2O2 accumulation, as well as the inhibition of catalase (CAT) genes expression. The mutant cat2-1, with increased H2O2 shows an accelerated senescence phenotype and decreased CHLI1 activity compared with the wild type. H2O2 inhibits CHLI1 activity by sulfenylating CHLI1 during leaf senescence. Consistent with this, the chli1 knockout mutant displays the same premature leaf senescence symptom as cat2-1, while overexpression of CHLI1 in cat2-1 can partially restore its early senescence phenotype. Taken together, these results illustrate that CAT2-mediated H2O2 accumulation during leaf senescence represses chlorophyll synthesis through sulfenylating CHLI1, and thus inhibits its activity, providing a new insight into the pivotal role of chlorophyll synthesis as a participant in orchestrating the leaf senescence.

Genetic diversity of naked barley accessions from the VIR collection for resistance to powdery mildew in the North-West Region of the Russian Federation

BACKGROUND: Barley is an important grain crop characterized by adaptability to various growing conditions. In recent years close attention has been paid to groups of naked barley which compared to covered barley is more susceptible to the influence of pathogens, that decreases the yield and quality of grain. Powdery mildew is the most harmful diseases of barley (caused by the biotrophic fungus Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal). The search for new promising sources of resistance to powdery mildew is necessary for barley breeding. AIM: The aim is studying the powdery mildew resistance of naked spring barley accessions from the world collection of VIR and identifying promising sources for economically valuable traits. MATERIALS AND METHODS: In field and laboratory conditions 271 of naked barley accessions of different ecological and geographical origin were studied for powdery mildew resistance and other economically valuable traits (lodging resistance, duration of the vegetative period, yield). The pathogen inoculum for laboratory work was represented by the north-western population of the pathogen. The resistance of barley samples was assessed in the field using the VIR point scale, in the laboratory using the E.B. Mains, S.M. Dietz point scale. The highly effective to powdery mildew resistance allele mlo11 was identified using PCR. RESULTS: The diversity of barley of different origins in resistance to powdery mildew is shown. 16 samples mainly from Europe, local forms from Ethiopia, Japan and India, were found to be resistant in field condition. According to the results of the assessment at the juvenile stage, 1 highly resistant sample k-31058 and 5 moderately resistant (k-20923, k-25801, k-27165, k-29911, k-30231) were identified. The naked forms k-20923 from Germany and k-29911 from Poland were resistant at all stages of plant development. The mlo11 allele was detected in the Arabische variety (k-20923) and heterogeneous pathogen resistance accessions k-20921, k-25793, k-27080, k-29440, k-29820. CONCLUSIONS: As a result of the work the sources of resistance to powdery mildew with a complex of economically valuable traits were identified, which can be involve in the breeding.

The microbiome of continuous wheat rotations: minor shifts in bacterial and archaeal communities but a source for functionally active plant-beneficial bacteria

Continuous wheat cropping often leads to yield decline, with suspected causes including increased pathogen susceptibility, decreased root growth, or low nutrient use efficiency, though the exact causes remain unknown. Here, we investigated soil and root-associated bacterial and archaeal communities in wheat under rotation versus continuous cultivation, monitored fungal pathogen presence, and used beneficial bacteria to mitigate Gaeumannomyces tritici (Ggt) symptoms in greenhouse experiments. Sampling was conducted at two field sites with different soil textures over two years and at two plant developmental stages, capturing site-specific and seasonal fluctuations. By cultivation, 767 bacterial isolates were screened for plant-beneficial traits. Amplicon sequencing revealed minor effects on community structure due to wheat rotational position but pronounced effects related to site, year, and microhabitat. Rhizoplane isolates with plant-beneficial traits were mainly Flavobacterium, Microbacterium, Pedobacter, Pseudomonas, Stenotrophomonas, and Variovorax. Continuous wheat rotations showed the highest proportion of bacteria with fungal antagonistic potential, indicating plant roots’ recruitment of beneficial bacteria. Introducing Ggt in the greenhouse experiment altered the bacterial and archaeal community, confirming field study results and demonstrating that applying beneficial bacteria early in the season at the seedling stage can control Ggt and improve plant growth. This study highlights the dynamic nature of wheat's below-ground bacterial and archaeal community, influenced by soil type, season, and microhabitat, with wheat rotation playing a minor role. In addition, it confirms the potential of selected Bacillus and Pseudomonas isolates, whether used individually or in consortia, for microbe-assisted mitigation of yield decline in intensive wheat production.

Transcriptional and hormonal profiling uncovers the interactions between plant developmental stages and RNA virus infection.

Arabidopsis thaliana is more susceptible to certain viruses during its later developmental stages. The differential responses and the mechanisms behind this development-dependent susceptibility to infection are still not fully understood. Here we explored the outcome of a viral infection at different host developmental stages by studying the response of A. thaliana to infection with turnip mosaic virus at three developmental stages: juvenile vegetative, bolting, and mature flowering plants. We found that infected plants at later stages downregulate cell wall biosynthetic genes and that this downregulation may be one factor facilitating viral spread and systemic infection. We also found that, despite being more susceptible to infection, infected mature flowering plants were more fertile (i.e. produce more viable seeds) than juvenile vegetative and bolting infected plants; that is, plants infected at the reproductive stage have greater fitness than plants infected at earlier developmental stages. Moreover, treatment of mature plants with salicylic acid increased resistance to infection at the cost of significantly reducing fertility. Together, these observations support a negative trade-off between viral susceptibility and plant fertility. Our findings point towards a development-dependent tolerance to infection.

SCREENING FLAX FOR IDENTIFYING CADMIUM TOLERANT CULTIVARS FOR GROWTH IN POLLUTED ENVIRONMENT

Toxic metal pollutants such as cadmium disrupt all stages of plant development. Flax (Linum usitatissimum L.) has demonstrated the ability to tolerate and accumulate high amounts of cadmium, making it suitable for phytoremediation. Within-species variability of cadmium tolerance and uptake needs to be examined in controled conditions to assess multiple beneficial, or even required traits for use in phytoremediation programs. In this study, 31 flax cultivars were screened for their ability to germinate and grow in the presence of a sublethal concentration of cadmium (20 mg.kg-1). Several parameters, including growth indicators and photosynthesis performance, were analyzed using principal component analysis. A subset of flax cultivars was then selected for measuring cadmium uptake using gamma spectrometry. The results showed that the cultivars Belinka, Diana, Laura, Marina, Flanders, and Jitka have comparable metal uptake and transfer to shoots. Among these, the cultivar Jitka proved to be the best candidate for phytoremediation, consistent with previous reports.

Assessing potato cultivars for resistance against late blight under high altitudinal conditions of North Western Himalayas

Different stages in plant development significantly influence the incidence of potato late blight disease in India. In the present study, the disease progression was monitored weekly in five selected potato cultivars viz. Kufri Jyoti, Kufri Giriraj, Gulmarg Special, Shalimar Potato-I, and Gurez local, during the 2018 and 2019 cropping seasons. The severity of potato late blight, caused by Phytophthora infestans, increased with fluctuations in weather variables. Calculations for the area under the disease progress curve (AUDPC) and the rate of disease spread (r-value) at different growth stages of potatoes were used to track disease progression. Notably, the Kufri Jyoti cultivar demonstrated resistance compared to the other cultivars, with lower severity values and susceptibility scale values (48.40%; 5.6 and 4.9) in 2018 and 2019, respectively. The average rate of disease progress was also lower in Kufri Jyoti (0.064 and 0.060) compared to the local cultivar (0.076 and 0.074) in 2018 and 2019, respectively. These observations indicate that weather parameters have a strong positive impact on the incidence of potato late blight.

Tailor-made green composts with suppressive properties against tracheofusariosis of wild rocket (Diplotaxis tenuifolia): Useful option for sustainable circular horticulture

Intensive cultivation systems of wild rocket (Diplotaxis tenuifolia) are threatened by the outbreak of tracheofusariosis caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. raphani. The disease is difficult to eradicate by curative methods due to the endophytic progression of the pathogen. Suppressive composts can help to prevent this adversity by providing organic matter carrying chemical and biological components that can directly and/or indirectly interfere with the plant-pathogen system. In this study, a collection of seven promising composts from different selected green feedstocks were investigated for their suppressive properties towards wild rocket Fusarium wilting. In planta assays identified two composts, obtained from olive tree pruning + plant and tomato residues, and from olive waste + straw + wool residues, with the highest levels of suppressiveness, influenced by both application dose and plant development stage. These active composts showed overlapping 13C NMR profiles as in vivo suppressiveness, being positively correlated with the organic aliphatic alkyl, O-Aryl and carboxyl C fractions and subsequently with the hydrophobic index and alkyl ratio, and negatively with the lignin ratio. On the contrary, although biotic compost components showed antifungal effects, biological properties did not correlate with in planta wilting suppression. Consistently, reflectance spectroscopy in the Vis-NIR-SWIR highlighted the clustering of samples depending on their feedstock and chemical composition. The results allow the hypothesis of a plant-mediated suppression mechanism. The composting of crop residues improves the circularity of horticulture and makes it possible to produce, from selected matrices, tailored compost towards the sustainable cultivation of wild rocket.

Genomic Organization and Expression Profiling of GOLDEN2-like Transcription Factor Genes in Eggplant and Their Role in Heat Stresses

GOLDEN2-like (GLK) transcription factor genes are involved in chloroplast biogenesis during all stages of plant growth and development, as well as in the response to biotic and abiotic stresses. However, little is known about this transcription factor family in eggplant. In this study, we identified 54 GLK genes in the eggplant genome (S. melongena L.) and classified them into seven groups (G1–G7). Structural analysis illustrated that the SmGLK proteins of specific groups are relatively conserved. Cis-acting elements indicated that these genes are likely to be involved in multiple responses stimulated by light, phytohormones, and abiotic stress. Collinear analysis indicated that expansion of the SmGLK gene family primarily occurred through segmental duplication. Tissue-specific expression analysis revealed that SmGLKs were preferentially expressed in leaves, fruits, and seeds. Further screening of SmGLK genes revealed their differential expression under various treatments. Notably, SmGLK18 was significantly responsive to multiple phytohormones and stress treatments, whereas SmGLK3 and SmGLK12 were highly induced by ABA, IAA, SA, and drought treatments. Our study provides new information on the eggplant GLK family systematically and comprehensively. For the first time, we propose that SmGLK18 may play a key role in improving heat resistance. This study provides valuable candidate gene resources for further functional research and will benefit eggplant molecular breeding.

Genome-wide association study provides new insight into the underlying mechanism of drought tolerance during seed germination stage in soybean

Drought is one of the major environmental issues that reduce crop yield. Seed germination is a crucial stage of plant development in all crop plants, including soybean. In soybean breeding, information about genetic mechanism of drought tolerance has great importance. However, at germination stage, there is relatively little knowledge on the genetic basis of soybean drought resistance. The objective of this work was to find the quantitative trait nucleotides (QTNs) linked to drought tolerance related three traits using a genome-wide association study (GWAS), viz., germination rate (GR), root length (RL), and whole seedling length (WSL), using germplasm population of 240 soybean PIs with 34,817 SNPs genotype data having MAF > 0.05. It was observed that heritability (H2) for GR, WSL, and RL across both environments (2020, and 2019) were high in the range of 0.76–0.99, showing that genetic factors play a vital role in drought tolerance as compared to environmental factors. A number of 23 and 27 QTNs were found to be linked to three traits using MLM and mrMLM, respectively. Three significant QTNs, qGR8-1, qWSL13-1, and qRL-8, were identified using both MLM and mrMLM methods among these QTNs. QTN8, located on chromosome 8 was consistently linked to two traits (GR and RL). The area (± 100 Kb) associated with this QTN was screened for drought tolerance based on gene annotation. Fifteen candidate genes were found by this screening. Based on the expression data, four candidate genes i.e. Glyma08g156800, Glyma08g160000, Glyma08g162700, and Glyma13g249600 were found to be linked to drought tolerance regulation in soybean. Hence, the current study provides evidence to understand the genetic constitution of drought tolerance during the germination stage and identified QTNs or genes could be utilized in molecular breeding to enhance the yield under drought stress.

Molecular and biochemical components associated with chilling tolerance in tomato: comparison of different developmental stages

The cultivated tomato, Solanum lycopersicum, is highly sensitive to cold stress (CS), resulting in significant losses during cultivation and postharvest fruit storage. Previously, we demonstrated the presence of substantial genetic variation in fruit chilling tolerance in a tomato recombinant inbred line (RIL) population derived from a cross between a chilling-sensitive tomato line and a chilling-tolerant accession of the wild species S. pimpinellifolium. Here, we investigated molecular and biochemical components associated with chilling tolerance in fruit and leaves, using contrasting groups of “chilling tolerant” and “chilling sensitive” RI lines. Transcriptomic analyses were conducted on fruit exposed to CS, and gene expressions and biochemical components were measured in fruit and leaves. The analyses revealed core responding genes specific to either the cold-tolerant or cold-sensitive RI lines, which were differentially regulated in similar fashion in both leaves and fruit within each group. These genes may be used as markers to determine tomato germplasm cold tolerance or sensitivity. This study demonstrated that tomato response to CS in different developmental stages, including seedling and postharvest fruit, might be mediated by common biological/genetic factors. Therefore, genetic selection for cold tolerance during early stages of plant development may lead to lines with greater postharvest fruit chilling tolerance.

Chlorophyll Degradation and its Physiological Function.

Research on chlorophyll degradation has progressed significantly in recent decades. In the 1990s, the structure of linear tetrapyrrole, which is unambiguously a chlorophyll degradation product, was determined. From the 2000s until the 2010s, the major enzymes involved in chlorophyll degradation were identified, and the pheophorbide a oxygenase/phyllobilin pathway was established. This degradation pathway encompasses several steps: (1) initial conversion of chlorophyll b to 7-hydroxymethyl chlorophyll a; (2) conversion of 7-hydroxymethyl chlorophyll a to chlorophyll a; (3) dechelation of chlorophyll a to pheophytin a; (4) dephytylation of pheophytin a to pheophorbide a; (5) opening of the macrocycle to yield a red chlorophyll catabolite; and (6) conversion of red chlorophyll catabolite to phyllobilins. This pathway converts potentially harmful chlorophyll into safe molecules of phyllobilins, which are stored in the central vacuole of terrestrial plants. The expression of chlorophyll-degrading enzymes is mediated by various transcription factors and influenced by light conditions, stress, and plant hormones. Chlorophyll degradation is differently regulated in different organs and developmental stages of plants. The initiation of chlorophyll degradation induces the further expression of chlorophyll-degrading enzymes, resulting in the acceleration of chlorophyll degradation. Chlorophyll degradation was initially considered the last reaction in senescence; however, chlorophyll degradation plays crucial roles in enhancing senescence, degrading chlorophyll-protein complexes, forming photosystem II, and maintaining seed quality. Therefore, controlling chlorophyll degradation has important agricultural applications.

Artificial neural networks as a tool for seasonal forecast of attack intensity of Spodoptera spp. in Bt soybean.

Soybean (Glycine max) is the world's most cultivated legume; currently, most of its varieties are Bt. Spodoptera spp. (Lepidoptera: Noctuidae) are important pests of soybean. An artificial neural network (ANN) is an artificial intelligence tool that can be used in the study of spatiotemporal dynamics of pest populations. Thus, this work aims to determine ANN to identify population regulation factors of Spodoptera spp. and predict its density in Bt soybean. For two years, the density of Spodoptera spp. caterpillars, predators, and parasitoids, climate data, and plant age was evaluated in commercial soybean fields. The selected ANN was the one with the weather data from 25 days before the pest's density evaluation. ANN forecasting and pest densities in soybean fields presented a correlation of 0.863. It was found that higher densities of the pest occurred in dry seasons, with less wind, higher atmospheric pressure and with increasing plant age. Pest density increased with the increase in temperature until this curve reached its maximum value. ANN forecasting and pest densities in soybean fields in different years, seasons, and stages of plant development were similar. Therefore, this ANN is promising to be implemented into integrated pest management programs in soybean fields.

Compositional Analysis of Cutin in Maize Leaves.

The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall-attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for Arabidopsis, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC-mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.