Preparation of Cellulose Nanoparticles from Foliage by Bio-Enzyme Methods

There are vast reserves of foliage in nature, which is an inexhaustible precious resource. In this study, the chemical components of five foliage types (pine needles, black locust tree leaves, bamboo leaves, elm leaves and poplar leaves) were analyzed, including cellulose content, hemicellulose content, and lignin content. The bio-enzymatic method was then used to prepare cellulose nanoparticles (CNPs) from these five kinds of leaves, and the prepared CNPs were analyzed using TEM, FTIR, FESEM, and XRD. The results showed that the content of hemicellulose in bamboo leaves was the highest, and the lignin content in the other four leaves was the highest. The cellulose content in the five kinds of foliage was arranged from large to small as pine needles (20.5%), bamboo leaves (19.5%), black locust leaves (18.0%), elm leaves (17.6%), and poplar leaves (15.5%). TEM images showed that the CNPs prepared by the five kinds of foliage reached the nanometer level in width and the micrometer level in length; therefore, the CNPs prepared in this study belonged to cellulose nanofibers (CNFs). The results of FTIR and XRD showed that CNFs prepared by the enzyme treatment exhibited a typical crystalline structure of cellulose II. The degree of crystallinity (DOC) of CNFs prepared from pine needle, poplar leaves, and bamboo leaves are 78.46%, 77.39%, and 81.51%, respectively. FESEM results showed that the CNFs prepared from pine needles, poplar leaves and bamboo leaves by enzymatic method presents a three-dimensional (3D) network structure, and their widths are 31 nm, 36 nm, and 37 nm, respectively. This study provides a meaningful reference for broadening the use of foliage types and improving their added value.

Diverse Understory Vegetation Alleviates Nitrogen Competition with Crop Trees in Poplar Plantations

Understory vegetation plays a crucial role in nutrient turnover and cycling in plantations, but it also competes for nutrients with crop trees when only a single species is present due to its specific nutrient requirements. However, it remains unclear whether this competition can be alleviated when the species richness of understory vegetation increases. In this study, we tested different gradients of understory vegetation species richness, including understory vegetation removal (UR), the retention of a single main understory vegetation species (RS), and the retention of natural diverse understory vegetation (RD) as part of a poplar (Populus deltoides ‘Nanlin-3804′) plantation, to study their effects on poplar growth, and to evaluate nitrogen (N) usage and how this was affected by the interactions between the poplar and understory vegetation. The results showed a generally lower periodic growth, and a significant decline in the foliar chlorophyll content and glutamine synthetase activity of poplar under treatment with RS and RD compared to those under UR treatment conducted in July 2019, which clearly indicated N competition between the understory vegetation and poplar trees. However, no significant difference was detected in the foliar chlorophyll content and glutamine synthetase activity of poplar under RD and RS treatment; only the nitrate reductase activity in poplar leaves under RD treatment declined significantly, by 22.25%, in June 2019. On the contrary, the diameter at breast height (DBH) of the poplar under RD treatment showed an increase of 34.69% from July to August 2019, compared with that under RS treatment. Furthermore, the increase in the species richness of understory vegetation resulted in an increase in the δ15N values in the poplar leaves, which was strongly regulated by the NH4+-N pool in the 10–20 cm soil layer, indicating the effective coordination of N utilization between poplar and understory vegetation when diversified understory plant species were present. These findings demonstrate the essential role of understory vegetation species diversity in alleviating N competition with crop trees, and provide guidance for understory vegetation management in poplar plantations.

Use of Black Poplar Leaves for the Biomonitoring of Air Pollution in an Urban Agglomeration

Trees are considered to be an effective tool for metal pollution biomonitoring. In the present study, the concentration of metals (Cu, Pb, Zn, Cd, Co, Ni, Fe, Mn, and Al) in black poplar leaves (Populus nigra L.), together with the concentration of PM10, PM2.5, PM1, and total suspended particles (TSP), was used for the air pollution biomonitoring in 12 sites from various areas of Cluj-Napoca city, Romania. The concentration of PM10 in the air was high, but their metal content was low. However, Cu, Pb, and Zn were moderately enriched, while Cd was highly enriched in PM10 due to anthropogenic sources. The average metal concentration in leaves decreased in the order Zn>>Fe>Mn>Al>Pb>Ni>Cu>Co>Cd and increased with the increase of PM10 concentration, indicating that poplar leaves are sensitive to air pollution. The principal component analysis indicated that traffic, waste burning, road dust resuspension, and soil contamination are the main anthropogenic sources of metals in poplar leaves. The results indicated that black poplar leaves are a suitable biomonitoring tool for metal pollution, in urban environments.

Genome-wide profiling of alternative splicing genes in hybrid poplar (P.alba×P.glandulosa cv.84K) leaves

Alternative splicing (AS) is a post-transcriptional process common in plants and essential for regulation of environmental fitness of plants. In the present study, we focus on the AS events in poplar leaves to understand their effects on plant growth and development. The hybrid poplar (P.alba×P.glandulosa cv.84K) leaves were collected for RNA extraction. The extracted RNA was sequenced using on an Illumina HiSeq™ 2000 platform. Using the Populus trichocarpa genome as the reference, a total of 3810 AS genes were identified (9225 AS events), which accounted for 13.51% of all the expressed genes. Intron retention was the most common AS event, accounting for 43.86% of all the AS events, followed by alternative 3′ splice sites (23.75%), alternative 5′ splice sites (23.71%), and exon skipping (8.68%). Chromosomes 10 had the most condensed AS events (33.67 events/Mb) and chromosome 19 had the least (12.42 events/Mb). Association analysis showed that AS in the poplar leaves was positively correlated with intron length, exon number, exon length, and gene expression level, and was negatively correlated with GC content. AS genes in the poplar leaves were associated mainly with inositol phosphate metabolism and phosphatidylinositol signaling system pathways that would be significant on wooden plant production.

An Automatic Method for Stomatal Pore Detection and Measurement in Microscope Images of Plant Leaf Based on a Convolutional Neural Network Model

Stomata are microscopic pores on the plant epidermis that regulate the water content and CO2 levels in leaves. Thus, they play an important role in plant growth and development. Currently, most of the common methods for the measurement of pore anatomy parameters involve manual measurement or semi-automatic analysis technology, which makes it difficult to achieve high-throughput and automated processing. This paper presents a method for the automatic segmentation and parameter calculation of stomatal pores in microscope images of plant leaves based on deep convolutional neural networks. The proposed method uses a type of convolutional neural network model (Mask R-CNN (region-based convolutional neural network)) to obtain the contour coordinates of the pore regions in microscope images of leaves. The anatomy parameters of pores are then obtained by ellipse fitting technology, and the quantitative analysis of pore parameters is implemented. Stomatal microscope image datasets for black poplar leaves were obtained using a large depth-of-field microscope observation system, the VHX-2000, from Keyence Corporation. The images used in the training, validation, and test sets were taken randomly from the datasets (562, 188, and 188 images, respectively). After 10-fold cross validation, the 188 test images were found to contain an average of 2278 pores (pore widths smaller than 0.34 μm (1.65 pixels) were considered to be closed stomata), and an average of 2201 pores were detected by our network with a detection accuracy of 96.6%, and the intersection of union (IoU) of the pores was 0.82. The segmentation results of 2201 stomatal pores of black poplar leaves showed that the average measurement accuracies of the (a) pore length, (b) pore width, (c) area, (d) eccentricity, and (e) degree of stomatal opening, with a ratio of width-to-maximum length of a stomatal pore, were (a) 94.66%, (b) 93.54%, (c) 90.73%, (d) 99.09%, and (e) 92.95%, respectively. The proposed stomatal pore detection and measurement method based on the Mask R-CNN can automatically measure the anatomy parameters of pores in plants, thus helping researchers to obtain accurate stomatal pore information for leaves in an efficient and simple way.

Dissecting MicroRNA–mRNA Regulatory Networks Underlying Sulfur Assimilation and Cadmium Accumulation in Poplar Leaves

The process of cadmium (Cd) accumulation and detoxification under different sulfur levels remains largely unknown in woody plants. To investigate the physiological and transcriptomic regulation mechanisms of poplars in response to different sulfate (S) supply levels and Cd exposure, we exposed Populus deltoides saplings to one of the low, moderate and high S levels together with either 0 or 50 µM Cd. Cd accumulation was decreased in low S-treated poplar leaves, and it tended to be increased in high S-supplied leaves under the Cd exposure condition. Sulfur nutrition was deficient in low S-supplied poplars, and it was improved in high S-treated leaves. Cd exposure resulted in lower sulfur level in the leaves supplied with moderate S, it exacerbated a Cd-induced sulfur decrease in low S-treated leaves and it caused a higher sulfur concentration in high S-supplied leaves. In line with the physiological changes, a number of mRNAs and microRNAs (miRNAs) involved in Cd accumulation and sulfur assimilation were identified and the miRNA–mRNA networks were dissected. In the networks, miR395 and miR399 members were identified as hub miRNAs and their targets were ATP sulfurylase 3 (ATPS3) and phosphate 2 (PHO2), respectively. These results suggest that Cd accumulation and sulfur assimilation are constrained by low and enhanced by high S supply, and Cd toxicity is aggravated by low and relieved by high S in poplar leaves, and that miRNA–mRNA regulatory networks play pivotal roles in sulfur-mediated Cd accumulation and detoxification in Cd-exposed poplars.