Suberin deficiency and its effect on the transport physiology of young poplar roots.

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

Apoplastic barriers of Populus × canescens roots in reaction to different cultivation conditions and abiotic stress treatments.

Populus is an important tree genus frequently cultivated for economical purposes. However, the high sensitivity of poplars towards water deficit, drought, and salt accumulation significantly affects plant productivity and limits biomass yield. Various cultivation and abiotic stress conditions have been described to significantly induce the formation of apoplastic barriers (Casparian bands and suberin lamellae) in roots of different monocotyledonous crop species. Thus, this study aimed to investigate to which degree the roots of the dicotyledonous gray poplar (Populus × canescens) react to a set of selected cultivation conditions (hydroponics, aeroponics, or soil) and abiotic stress treatments (abscisic acid, oxygen deficiency) because a differing stress response could potentially help in explaining the observed higher stress susceptibility. The apoplastic barriers of poplar roots cultivated in different environments were analyzed by means of histochemistry and gas chromatography and compared to the available literature on monocotyledonous crop species. Overall, dicotyledonous poplar roots showed only a remarkably low induction or enhancement of apoplastic barriers in response to the different cultivation conditions and abiotic stress treatments. The genetic optimization (e.g., overexpression of biosynthesis key genes) of the apoplastic barrier development in poplar roots might result in more stress-tolerant cultivars in the future.

Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar.

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Cutinized and suberized barriers in leaves and roots: Similarities and differences.

Anatomical, histochemical, chemical, and biosynthetic similarities and differences of cutinized and suberized plant cell walls are presented and reviewed in brief. Based on this, the functional properties of cutinized and suberized plant cell walls acting as transport barriers are compared and discussed in more detail. This is of general importance because fundamental misconceptions about relationships in plant-environment water relations are commonly encountered in the scientific literature. It will be shown here, that cuticles represent highly efficient apoplastic transport barriers significantly reducing the diffusion of water and dissolved compounds. The transport barrier of cuticles is mainly established by the deposition of cuticular waxes. Upon wax extraction, with the cutin polymer remaining, cuticular permeability for water and dissolved non-ionized and lipophilic solutes are increasing by 2-3 orders of magnitude, whereas polar and charged substances (e.g., nutrient ions) are only weakly affected (2- to 3-fold increases in permeability). Suberized apoplastic barriers without the deposition of wax are at least as permeable as the cutin polymer matrix without waxes and hardly offer any resistance to the free movement of water. Only upon the deposition of significant amounts of wax, as it is the case with suberized periderms exposed to the atmosphere, an efficient transport barrier for water can be established by suberized cell walls. Comparing the driving forces (gradients between water potentials inside leaves and roots and the surrounding environment) for water loss acting on leaves and roots, it is shown that leaves must have a genetically pre-defined highly efficient transpiration barrier fairly independent from rapidly changing environmental influences. Roots, in most conditions facing a soil environment with relative humidities very close to 100%, are orders of magnitude more permeable to water than leaf cuticles. Upon desiccation, the permanent wilting point of plants is defined as -1.5 MPa, which still corresponds to nearly 99% relative humidity in soil. Thus, the main reason for plant water stress leading to dehydration is the inability of root tissues to decrease their internal water potential to values more negative than -1.5 MPa and not the lack of a transport barrier for water in roots and leaves. Taken together, the commonly mentioned concepts that a drought-induced increase of cuticular wax or root suberin considerably strengthens the apoplastic leaf or root transport barriers and thus aids in water conservation appears highly questionable.

Populus × canescens root suberization in reaction to osmotic and salt stress is limited to the developing younger root tip region.

Populus is a valuable and fast-growing tree species commonly cultivated for economic and scientific purposes. But most of the poplar species are sensitive to drought and salt stress. Thus, we compared the physiological effects of osmotic stress (PEG8000) and salt treatment (NaCl) on poplar roots to identify potential strategies for future breeding or genetic engineering approaches. We investigated root anatomy using epifluorescence microscopy, changes in root suberin composition and amount using gas chromatography, transcriptional reprogramming using RNA sequencing, and modifications of root transport physiology using a pressure chamber. Poplar roots reacted to the imposed stress conditions, especially in the developing younger root tip region, with remarkable differences between both types of stress. Overall, the increase in suberin content was surprisingly small, but the expression of key suberin biosynthesis genes was strongly induced. Significant reductions of the radial water transport in roots were only observed for the osmotic and not the hydrostatic hydraulic conductivity. Our data indicate that the genetic enhancement of root suberization processes in poplar might be a promising target to convey increased tolerance, especially against toxic sodium chloride.

Genome-wide analysis of long non-coding RNAs in shoot apical meristem and vascular cambium in Populus tomentosa.

Shoot apical and lateral meristems play essential roles in the formation and development of primary and secondary growth in plants. A delicate regulatory mechanism is needed to maintain homeostatic balance between the primary and secondary growth, as well as the self-renewal of meristems with the rate of cell division and differentiation of new meristems. However, little is known about the roles of long non-coding RNAs (lncRNAs) in the regulation of maintenance and differentiation of primary and secondary growth in Populus, especially in the cambium division and differentiation into secondary xylem. Here, 1298 lncRNAs were identified both in the apical meristem and vascular cambium, with 80 lncRNAs being expressed only in shoot apical meristem and 45 only in vascular cambium. There are 410 differentially expressed lncRNAs in shoot apical meristem and vascular cambium, among which 271 lncRNAs were up-regulated and 139 were down-regulated in cambium. The GO enrichment analysis revealed that differentially expressed lncRNAs mainly influenced the expression of lncRNAs related to the ribosome pathway, plant hormone signal pathway and photosynthesis pathway. The differentially expressed lncRNAs mainly target mRNA through cis-regulation in the vascular cambium. In addition, six key lncRNAs and also their significantly upregulated target genes were identified. Theses target genes are involved in plant secondary metabolites, cellulose and lignin synthesis, hormone and signal transduction. In addition, six key lncRNAs were identified, their significantly upregulated target genes are related to plant secondary metabolites, cellulose and lignin synthesis, hormone and signal transduction. Investigating lncRNA-mRNA interactions, we further found some genes that may be related to the development of vascular cambium, such as domain-containing transcription factors, cellulose synthesis genes, calcium dependent protein kinase 2, cytokinin receptor 1, glycosyl transferase and polyphenol oxidase. Our findings provide new insights into the lncRNA-mRNA networks in the development of vascular cambium of secondary growth in Populus.

Increased cuticular wax deposition does not change residual foliar transpiration.

The effect of contrasting environmental growth conditions (in vitro tissue culture, ex vitro acclimatisation, climate chamber, greenhouse and outdoor) on leaf development, cuticular wax composition, and foliar transpiration of detached leaves of the Populus × canescens clone 84 K were investigated. Our results show that total amounts of cuticular wax increased more than 10-fold when cultivated in different growth conditions, whereas qualitative wax composition did not change. With exception of plants directly taken from tissue culture showing rapid dehydration, rates of water loss (residual foliar transpiration) of intact but detached leaves were constant and independent from growth conditions and thus independent from increasing wax amounts. Since cuticular transpiration measured with isolated astomatous P. × canescens cuticles was identical to residual foliar transpiration rates of detached leaves, our results confirm that cuticular transpiration of P. × canescens leaves can be predicted with high accuracy from residual transpiration of detached leaves after stomatal closure. Our results convincingly show that more than 10-fold increased wax amounts in P. × canescens cuticles do not lead to decreased rates of residual (cuticular) transpiration.

Hydroponic cultivation conditions allowing the reproducible investigation of poplar root suberization and water transport.

BACKGROUND: With increasing joint research cooperation on national and international levels, there is a high need for harmonized and reproducible cultivation conditions and experimental protocols in order to ensure the best comparability and reliability of acquired data. As a result, not only comparisons of findings of different laboratories working with the same species but also of entirely different species would be facilitated. As Populus is becoming an increasingly important genus in modern science and agroforestry, the integration of findings with previously gained knowledge of other crop species is of high significance. RESULTS: To ease and ensure the comparability of investigations of root suberization and water transport, on a high degree of methodological reproducibility, we set up a hydroponics-based experimental pipeline. This includes plant cultivation, root histochemistry, analytical investigation, and root water transport measurement. A 5-week-long hydroponic cultivation period including an optional final week of stress application resulted in a highly consistent poplar root development. The poplar roots were of conical geometry and exhibited a typical Casparian band development with subsequent continuously increasing suberization of the endodermis. Poplar root suberin was composed of the most frequently described suberin substance classes, but also high amounts of benzoic acid derivatives could be identified. Root transport physiology experiments revealed that poplar roots in this developmental stage have a two- to tenfold higher hydrostatic than osmotic hydraulic conductivity. Lastly, the hydroponic cultivation allowed the application of gradually defined osmotic stress conditions illustrating the precise adjustability of hydroponic experiments as well as the previously reported sensitivity of poplar plants to water deficits. CONCLUSIONS: By maintaining a high degree of harmonization, we were able to compare our results to previously published data on root suberization and water transport of barley and other crop species. Regarding hydroponic poplar cultivation, we enabled high reliability, reproducibility, and comparability for future experiments. In contrast to abiotic stress conditions applied during axenic tissue culture cultivation, this experimental pipeline offers great advantages including the growth of roots in the dark, easy access to root systems before, during, and after stress conditions, and the more accurate definition of the developmental stages of the roots.

Transcriptomic and epigenomic remodeling occurs during vascular cambium periodicity in Populus tomentosa.

Trees in temperate regions exhibit evident seasonal patterns, which play vital roles in their growth and development. The activity of cambial stem cells is the basis for regulating the quantity and quality of wood, which has received considerable attention. However, the underlying mechanisms of these processes have not been fully elucidated. Here we performed a comprehensive analysis of morphological observations, transcriptome profiles, the DNA methylome, and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation. Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium. Furthermore, we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction, cell division and expansion, and cell wall biosynthesis. In addition, we identified 235 known miRNAs and 125 novel miRNAs. Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs. Moreover, global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression, whereas CHG exhibited the opposite trend in the downstream region. Most importantly, we observed that the number of CHH differentially methylated region (DMR) changes was the greatest during cambium periodicity. Intriguingly, the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interactions during vascular cambium development. These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.

Tissue-specific and maturity-dependent distribution of pyridine alkaloids in Areca triandra.

Areca nuts (seeds of Areca catechu L.) are a traditional and popular masticatory in India, Bangladesh, Malaysia, certain parts of China, and some other countries. Four related pyridine alkaloids (arecoline, arecaidine, guvacoline, and guvacine) are considered being the main functional ingredients in areca nut. Until now, A. catechu is the only known species producing these alkaloids in the Arecaceae family. In the present study, we investigated alkaloid contents in 12 Arecaceae species and found that only Areca triandra Roxb. contained these pyridine alkaloids. We further analyzed in more detail tissue-specific and development-related distribution of these alkaloids in leaves, male and female flowers and fruits in different stages of maturity in A. triandra by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. Results revealed that the alkaloids were most abundant in young leaves, the pericarp of ripe fruits and the endosperm of unripe fruits in developmental stage 2. Abundance of the 4 different alkaloids in A. triandra fruits varied during maturation. Pericarps of ripe fruits had the highest arecaidine concentration (4.45 mg g-1) and the lowest guvacoline concentration (0.0175 mg g-1), whereas the endosperm of unripe fruits of developmental stage 2 contained the highest guvacoline concentration (3.39 mg g-1) and the lowest guvacine concentration (0.245 mg g-1). We conclude that A. triandra is useful in future as a further valuable source of Areca alkaloids.