Micromorphology of endemic Centaurea glaberrima subsp. divergens (Asteraceae)

In this study, the micromorphology of the vegetative and reproductive structures of the endemic Centaurea glaberrima Tausch subsp. divergens (Vis.) Hayek (Asteraceae), using scanning electron microscope (SEM), is presented for the first time. Uniseriate whip-like non-glandular and biseriate glandular trichomes are found on the surface of all aboveground parts (stem, leaves, peduncles, involucral bract). On the adaxial leaf epidermis ribbed thickenings (striation pattern) of outer periclinal cell walls, slightly curved anticlinal cell walls and anomocytic stomata are noticed. Rugose abaxial surface with thorny protuberances of the involucral bract is documented. Corolla is glabrous with longitudinally parallel epidermal cells with distinct straight outline. Isopolar, radially symmetric and tricolporate microechinate pollen grains are seen. Short stylar hairs, without cuticular striations, are present along the outer sides of the style, while the inner sides (abaxial surface) constitute the papillate stigmatic surface. Microcharacters found in cypsela are as follows: slightly ribbed body; rotund base; lateral and concave insertion; short, unicellular curly acute trichomes; smooth epidermis; fine-sulcate ornamentation; rod shaped epidermal cells with short, obtuse end walls and straight anticlinal walls; poorly developed minutely dentate pericarp rim; and dimorphic pappus with bristles of different length and morphology, with pinnules restricted to the margins of the bristles. The results obtained contribute to knowledge about the micromorphology of the studied endemic species and provide features for its better identification. The taxonomic significance of the analyzed characters is discussed. Some well defined microcharacters of the studied species might have taxonomic value

Identification of the ploidy level of plants in grape breeding

Полиплоидные формы обращают на себя внимание своими положительными свойствами, одним из которых является увеличение по сравнению с диплоидными сортами размеров хозяйственно ценных органов. Значительное количество работ по полиплоидии растений в последнее время обусловлено развитием аналитических методов, таких как современные способы приготовления цитогенетических препаратов, цифровая микроскопия, проточная цитометрия, ПЦР-анализ. В настоящее время для получения полиплоидных форм растений используют системы культуры ткани. Успех индукции полиплоидии зависит от различных факторов: состава питательной среды, антимитотического агента, типа эксплантов, времени воздействия и концентрации веществ. В программах создания полиплоидных форм растений проводят исследования с использованием прямого подсчета хромосом, проточной цитометрии, ПЦР-анализа, а также косвенных методов изучения морфологических особенностей объектов. Методы изучения структуры эпидермиса листа являются простыми, быстрыми, неразрушающими и не требующими дорогих реагентов или оборудования. В качестве морфологических индикаторов плоидности обычно используют параметры устьиц (частота устьиц, размеры замыкающих клеток и количество хлоропластов в устьицах). Предлагаются простые протоколы прямого и косвенного методов анализа плоидности винограда. Наиболее успешными работами в области изучения плоидности растений можно считать исследования комплексные. Косвенные методы анализа следует использовать для массового скрининга исходной выборки, прямые методы - для точного изучения генома отобранных растений. Изучение морфологических особенностей эпидермиса листьев может быть использовано в селекционных программах создания виноградных полиплоидов. Исследование дает рациональное обоснование дальнейшей работы по анализу цитогенетических и морфологических особенностей полиплоидных растений винограда. Polyploid forms attract attention for their positive properties, one of which is an increase in the size of economically valuable organs compared to diploid varieties. A significant number of works on plant polyploidy in recent years is due to the development of analytical methods, such as modern methods for the preparation of microslides, digital microscopy, flow cytometry, PCR- analysis. Tissue culture systems are currently used to obtain polyploid forms of plants. The success of polyploidy induction depends on various factors, such as composition of the nutrient medium, antimitotic agent, type of explants, time of exposure, and concentration of substances. In programs for creating polyploid forms of plants, the research is carried out using direct chromosome counting, flow cytometry, PCR-analysis, as well as indirect methods for studying morphological characteristics of objects. Methods for study the structure of leaf epidermis are simple, fast, non-destructive and not requiring expensive reagents or equipment. Stomatal parameters (stomatal density, guard cell size, and the number of chloroplasts in stoma) are commonly used as morphological indicators of ploidy. Simple protocols of direct and indirect methods of ploidy analysis for grapes are proposed. Complex research can be considered as the most successful in the field of plant ploidy studies. Indirect methods of analysis should be used for mass screening of the initial sample, direct methods - for precise study of the genome of selected plants. The study of morphological characteristics of leaf epidermis can be used in breeding programs for the creation of grape polyploids. The research provides a rational basis for further work on the analysis of cytogenetic and morphological features of polyploid grape plants.

Activation of 1-Aminocyclopropane-1-Carboxylic Acid Synthases Sets Stomatal Density and Clustered Ratio on Leaf Epidermis of Arabidopsis in Response to Drought

The adjustment of stomatal density and clustered ratio on the epidermis is the important strategy for plants to respond to drought, because the stoma-based water loss is directly related to plant growth and survival under drought conditions. But the relevant adjustment mechanism still needs to be explored. 1-Aminocyclopropane-1-carboxylate (ACC) is disclosed to promote stomatal development, while in vivo ACC levels depend on activation of ACC synthase (ACS) family members. Based on the findings of ACS expression involving in drought response and several ACS activity inhibitors reducing stomatal density and cluster in drought response, here we examined how ACS activation is involved in the establishment of stomatal density and cluster on the epidermis under drought conditions. Preliminary data indicated that activation of ACS2 and/or ACS6 (ACS2/6) increased stomatal density and clustered ratio on the Arabidopsis leaf epidermis by accumulating ACC under moderate drought, and raised the survival risk of seedlings under escalated drought. Further exploration indicated that, in Arabidopsis seedlings stressed by drought, the transcription factor SPEECHLESS (SPCH), the initiator of stomatal development, activates ACS2/6 expression and ACC production; and that ACC accumulation induces Ca2+ deficiency in stomatal lineage; this deficiency inactivates a subtilisin-like protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) by stabilizing the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression, resulting in an increases of stomatal density and cluster ratio on the leaf epidermis. This work provides a novel evidence that ACS2/6 activation plays a key role in the establishment of stomatal density and cluster on the leaf epidermis of Arabidopsis in response to drought.

Analysis of Wheat Wax Regulation Mechanism by Liposome and Transcriptome

As a barrier for plants to contact with the outside world, epidermal wax plays an important role in resisting biotic and abiotic stresses. In this study, we analyzed the effect of wax content on leaf permeability by measuring the wax loss rate in the leaf. To further clarify the wax composition of the wheat epidermis and its molecular regulation mechanism, we applied untargeted lipidomic and transcriptome analysis on the leaf epidermis wax of Jimai 22 low-wax mutant (waxless) and multi-wax mutant (waxy). Our research showed that the mutant waxy has a slow loss rate, which can maintain higher leaf water content. 31 lipid subclasses and 1,367 lipid molecules were identified. By analyzing the wax differences of the two mutants, we found that the main lipid components of leaf epidermis wax in Jimai 22 were WE (C19-C50), DG (C27-C53), MG (C31-C35), and OAHFA (C31-C52). Carbon chain length analysis showed that, in wheat epidermis wax, WE was dominated by C44 molecules, DG was mainly concentrated in C47, C45, C37, and C31 molecules, C48 played a leading role in OAHFA, and C35 and C31 played a major role in MG. Among them, DG, MG, and OAHFA were detected in wheat leaf wax for the first time, and they were closely related to stress resistance. Compared with the waxy, 6,840 DEGs were detected in the mutant waxless, 3,181 DEGs were upregulated, and 3,659 DEGs were downregulated. The metabolic pattern of main waxy components in the wheat epidermis was constructed according to KEGG metabolic pathway and 46 related genes were screened, including KSC, TER, FAR, WSD1, CER1, MAH1, ALDH7A1, CYP704B1, ACOT1_2_4, CYP86, MGLL, GPAT, ALDH, DPP1, dgkA, plsC, and E2.3.1.158 related genes. The screened wax-related genes were confirmed to be highly reliable by qRT-PCR. In addition, we found TER gene TraesCS6B03G1132900LC in wheat mutant waxless leaves for the first time, which inhibited the synthesis of long-chain acyl-CoA (n+2) by downregulating its expression. These results provide valuable reference information for further study of wheat epidermis wax heredity and molecular regulation.

Differential Occurrence of Cuticular Wax and Its Role In Leaf Tissues of Three Edible Aroids of Northeast India

Localization of cuticular wax (CW) on the leaf epidermis and its interaction with physiological mechanisms of three edible aroids, Alocasia, Colocasia, and Xanthosoma, were assessed. Scanning electron microscopy depicted the occurrence of CW in the leaf tissues, which was higher in Colocasia (10.61 mg dm-2) and Xanthosoma (11.36 mg dm-2) than in Alocasia (1.36 mg dm-2). Higher CW in Colocasia and Xanthosoma strengthened leaf epidermis and improved the physiological processes compared to Alocasia. CW acted as a protecting barrier against deleterious solar radiation in terms of sun protection factor (SPF). The glossy appearance of wax crystals in the Alocasia leaf cuticles resulted in higher SPF. The occurrence of CW was directly related to leaf chlorophyll stability, moisture retention ability, and cellular membrane integrity in the leaf tissues. Colocasia exhibited superhydrophobic properties with higher static contact angle (CA) >150o than hydrophobic Xanthosoma, and Alocasia with CA ranged between 99.0o to 128.7o. Colocasia CW highly influenced the qualitative and protective mechanisms of the leaf. Aroids are the cheapest sources of edible CW among the terrestrial plants, which could be used in food, agricultural and industrial applications.

A simple method for the application of exogenous phytohormones to the grass leaf base protodermal zone to improve grass leaf epidermis development research

Background The leaf epidermis functions to prevent the loss of water and reduce gas exchange. As an interface between the plant and its external environment, it helps prevent damage, making it an attractive system for studying cell fate and development. In monocotyledons, the leaf epidermis grows from the basal meristem that contains protodermal cells. Leaf protoderm zone is covered by the leaf sheath or coleoptile in maize and wheat, preventing traditional exogenous phytohormone application methods, such as directly spraying on the leaf surface or indirectly via culture media, from reaching the protoderm areas directly. The lack of a suitable application method limits research on the effect of phytohormone on the development of grass epidermis. Results Here, we describe a direct and straightforward method to apply exogenous phytohormones to the leaf protoderms of maize and wheat. We used the auxin analogs 2,4-D and cytokinin analogs 6-BA to test the system. After 2,4-D treatment, the asymmetrical division events and initial stomata development were decreased, and the subsidiary cells were induced in maize, the number of GMC (guard mother cell), SMC (subsidiary mother cell) and young stomata were increased in wheat, and the size of the epidermal cells increased after 6-BA treatment in maize. Thus, the method is suitable for the application of phytohormone to the grass leaf protodermal areas. Conclusions The method to apply hormones to the mesocotyls of maize and wheat seedlings is simple and direct. Only a small amount of externally applied substances are needed to complete the procedure in this method. The entire experimental process lasts for ten days generally, and it is easy to evaluate the phytohormones’ effect on the epidermis development.

Leaf epidermal micromorphology in Aspidistra (Asparagaceae): diversity and taxonomic significance

Micromorphological characters of leaf epidermis were investigated in 69 species of Aspidistra using scanning electron microscopy. Sculpture of epidermis varies from smooth to verrucose and rugose in the genus. The abaxial epidermis of some species bears papillae, whereas the adaxial surface uniformly lacks the papillae. Sculpture type of epidermis and density of papillae are generally found to be stable characters at a species level. The infraspecific variation of epidermis sculpture, where present, ranges from smooth to verrucose or from verrucose to rugose. Micromorphological characters of leaf epidermis are shown to have potential taxonomic significance in Aspidistra; in combination with the type of shoot structure, they allow to subdivide the species into 13 groups. The groups are largely incongruent with floral morphological traits. An identification key to the studied species of Aspidistra based on vegetative characters (gross leaf and shoot morphology and characters of leaf epidermis) is presented.

Atlas of leaf surface micromorphology in Aloe L. (Asphodelaceae) from the Horn of Africa region

The confident identification to species rank of fragmentary and sterile plant material is often challenged by the absence of diagnostic characters, which are present in intact specimens, reproductive parts, and plants in habitat. Here, we consider leaf surface micromorphology for the identification of the genus Aloe in the Horn of Africa region. Primary and secondary sculpturing of the leaf epidermis and stomata were characterised from SEM micrographs of 35 taxa representing 31 species of Aloe (Asphodelaceae subfam. Alooideae). Detailed comparison revealed that leaf surface characters are conserved between species and within-species variation is modest. Closely related taxa in the Aloe adigratana—A. camperi—A. sinana species complex could be distinguished using leaf surface micromorphology alone. These characters also guide species delimitation; in the species complex including A. schoelleri and A. steudneri, a narrow circumscription is supported, whereas with A. ankoberensis and A. pulcherrima, a wider circumscription merits consideration. The observed trait combinations are characteristic of plants in xeric environments, with the most notable feature being stomata that are most deeply sunken in species in more arid habitats. Our findings support the use of comparative study of micromorphological leaf surface characters for species identification and taxonomy in the genus Aloe.

A Deep Learning-Based Method for Automatic Assessment of Stomatal Index in Wheat Microscopic Images of Leaf Epidermis

The stomatal index of the leaf is the ratio of the number of stomata to the total number of stomata and epidermal cells. Comparing with the stomatal density, the stomatal index is relatively constant in environmental conditions and the age of the leaf and, therefore, of diagnostic characteristics for a given genotype or species. Traditional assessment methods involve manual counting of the number of stomata and epidermal cells in microphotographs, which is labor-intensive and time-consuming. Although several automatic measurement algorithms of stomatal density have been proposed, no stomatal index pipelines are currently available. The main aim of this research is to develop an automated stomatal index measurement pipeline. The proposed method employed Faster regions with convolutional neural networks (R-CNN) and U-Net and image-processing techniques to count stomata and epidermal cells, and subsequently calculate the stomatal index. To improve the labeling speed, a semi-automatic strategy was employed for epidermal cell annotation in each micrograph. Benchmarking the pipeline on 1,000 microscopic images of leaf epidermis in the wheat dataset (Triticum aestivum L.), the average counting accuracies of 98.03 and 95.03% for stomata and epidermal cells, respectively, and the final measurement accuracy of the stomatal index of 95.35% was achieved. R2 values between automatic and manual measurement of stomata, epidermal cells, and stomatal index were 0.995, 0.983, and 0.895, respectively. The average running time (ART) for the entire pipeline could be as short as 0.32 s per microphotograph. The proposed pipeline also achieved a good transferability on the other families of the plant using transfer learning, with the mean counting accuracies of 94.36 and 91.13% for stomata and epidermal cells and the stomatal index accuracy of 89.38% in seven families of the plant. The pipeline is an automatic, rapid, and accurate tool for the stomatal index measurement, enabling high-throughput phenotyping, and facilitating further understanding of the stomatal and epidermal development for the plant physiology community. To the best of our knowledge, this is the first deep learning-based microphotograph analysis pipeline for stomatal index assessment.