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.

Abscisic acid is required for exodermal suberization to form a barrier to radial oxygen loss in the adventitious roots of rice (Oryza sativa).

To acclimate to waterlogged conditions, wetland plants form a barrier to radial oxygen loss (ROL) that can enhance oxygen transport to the root apex. We hypothesized that one or more hormones are involved in the induction of the barrier and searched for such hormones in rice. We previously identified 98 genes that were tissue-specifically upregulated during ROL barrier formation in rice. The RiceXPro database showed that most of these genes were highly enhanced by exogenous abscisic acid (ABA). We then examined the effect of ABA on ROL barrier formation by using an ABA biosynthesis inhibitor (fluridone, FLU), by applying exogenous ABA and by examining a mutant with a defective ABA biosynthesis gene (osaba1). FLU suppressed barrier formation in a stagnant solution that mimics waterlogged soil. Under aerobic conditions, rice does not naturally form a barrier, but 24 h of ABA treatment induced barrier formation. osaba1 did not form a barrier under stagnant conditions, but the application of ABA rescued the barrier. In parallel with ROL barrier formation, suberin lamellae formed in the exodermis. These findings strongly suggest that ABA is an inducer of suberin lamellae formation in the exodermis, resulting in an ROL barrier formation in rice.

Cuticular transpiration is not affected by enhanced wax and cutin amounts in response to osmotic stress in barley.

The plant cuticle, which covers all aerial parts of plants in their primary developmental stage, is the major barrier against water loss from leaves. Accumulation of cutin and waxes has often been linked to drought tolerance. Here we investigated whether cutin and waxes play a role in the drought adaption of barley mimicked by osmotic stress acting on roots. We compared the cuticle properties of cultivated barley (Hordeum vulgare spp. vulgare) with wild barley (Hordeum vulgare spp. spontaneum), and tested whether wax and cutin composition or amount and cuticular transpiration could be future breeding targets for more drought-tolerant barley lines. In response to osmotic stress, accumulation of wax crystals was observed. This coincides with an increased wax and cutin gene expression and a total increase of wax and cutin amounts in leaves, which seems to be a general response triggered through root shoot signalling. Stomatal conductance decreased fast and significantly, whereas cuticular conductance remained unaffected in both wild and cultivated barley. The often-made conclusion that higher amounts of wax and cutin necessarily reduce cuticular transpiration and thus enhance drought tolerance is not always straightforward. To prevent water loss, stomatal regulation under water stress is much more important than regulation or adaptation of cuticular transpiration in response to drought.

Surface wax esters contribute to drought tolerance in Arabidopsis.

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression, suberization, and hydraulic conductivity.

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.

Suberized transport barriers in plant roots: the effect of silicon.

Plant roots are the major organs that take up water and dissolved nutrients. It has been widely shown that apoplastic barriers such as Casparian bands and suberin lamellae in the endo- and exodermis of roots have an important effect on regulating radial water and nutrient transport. Furthermore, it has been described that silicon can promote plant growth and survival under different conditions. However, the potential effects of silicon on the formation and structure of apoplastic barriers are controversial. A delayed as well as an enhanced suberization of root apoplastic barriers with silicon has been described in the literature. Here we review the effects of silicon on the formation of suberized apoplastic barriers in roots, and present results of the effect of silicon treatment on the formation of endodermal suberized barriers on barley seminal roots under control conditions and when exposed to osmotic stress. Chemical analysis confirmed that osmotic stress enhanced barley root suberization. While a supplementation with silicon in both, control conditions and osmotic stress, did not enhanced barley root suberization. These results suggest that enhanced stress tolerance of plants after silicon treatment is due to other responses.

Osmotic stress enhances suberization of apoplastic barriers in barley seminal roots: analysis of chemical, transcriptomic and physiological responses.

Barley (Hordeum vulgare) is more drought tolerant than other cereals, thus making it an excellent model for the study of the chemical, transcriptomic and physiological effects of water deficit. Roots are the first organ to sense soil water deficit. Therefore, we studied the response of barley seminal roots to different water potentials induced by polyethylene glycol (PEG) 8000. We investigated changes in anatomical parameters by histochemistry and microscopy, quantitative and qualitative changes in suberin composition by analytical chemistry, transcript changes by RNA-sequencing (RNA-Seq), and the radial water and solute movement of roots using a root pressure probe. In response to osmotic stress, genes in the suberin biosynthesis pathway were upregulated that correlated with increased suberin amounts in the endodermis and an overall reduction in hydraulic conductivity (Lpr ). In parallel, transcriptomic data indicated no or only weak effects of osmotic stress on aquaporin expression. These results indicate that osmotic stress enhances cell wall suberization and markedly reduces Lpr of the apoplastic pathway, whereas Lpr of the cell-to-cell pathway is not altered. Thus, the sealed apoplast markedly reduces the uncontrolled backflow of water from the root to the medium, whilst keeping constant water flow through the highly regulated cell-to-cell path.

Night-time transpiration in barley (Hordeum vulgare) facilitates respiratory carbon dioxide release and is regulated during salt stress.

Background and Aims: Night-time transpiration accounts for a considerable amount of water loss in crop plants. Despite this, there remain many questions concerning night-time transpiration - its biological function, regulation and response to stresses such as salinity. The aim of the present study was to address these questions on 14- to 18-d-old, hydroponically grown barley plants. Methods: Plants were either stressed for the last 4-7 d prior to, and during subsequent continuous (24 h), diurnal gravimetric transpiration analyses; or subjected to salt stress just before analyses; or stressed for 4-7 d and then transferred to control medium before analyses. The idea behind this experimental setup was to distinguish between a longer- (cuticle, stomata) and shorter-term (stomata) response of transpiration to treatments. Cuticular conductance was assessed through residual transpiration measurements in detached leaves. Cuticle wax load and dark respiration rate of leaves were determined. Leaf conductance to CO2 was calculated. Key Results: Night-time and daytime transpiration rates were highly, and positively, correlated with each other, across all treatments. Night-time transpiration rates accounted for 9-17 % of daytime rates (average: 13.8 %). Despite minor changes in the ratio of night- to daytime transpiration rates, the contribution of cuticular and stomatal conductance to leaf (epidermal) conductance to water vapour differed considerably between treatments. Salt stress did not affect cuticle wax load. The conductance for CO2 of the cuticle was insufficient to support rates of dark respiratory CO2 release. Conclusions: The main biological function of night-time transpiration is the release of respiratory CO2 from leaves. Night-time transpiration is regulated in the short and long term, also under salt stress. Stomata play a key role in this process. We propose to refer, in analogy to water use efficiency (WUE) during the day, to a CO2 release efficiency ('CORE') during the night.

The composite water and solute transport of barley (Hordeum vulgare) roots: effect of suberized barriers.

Background and Aims: Roots have complex anatomical structures, and certain localized cell layers develop suberized apoplastic barriers. The size and tightness of these barriers depend on the growth conditions and on the age of the root. Such complex anatomical structures result in a composite water and solute transport in roots. Methods: Development of apoplastic barriers along barley seminal roots was detected using various staining methods, and the suberin amounts in the apical and basal zones were analysed using gas chromatography-mass spectometry (GC-MS). The hydraulic conductivity of roots ( Lp r ) and of cortical cells ( Lp c ) was measured using root and cell pressure probes. Key Results: When grown in hydroponics, barley roots did not form an exodermis, even at their basal zones. However, they developed an endodermis. Endodermal Casparian bands first appeared as 'dots' as early as at 20 mm from the apex, whereas a patchy suberin lamellae appeared at 60 mm. The endodermal suberin accounted for the total suberin of the roots. The absolute amount in the basal zone was significantly higher than in the apical zone, which was inversely proportional to the Lp r . Comparison of Lp r and Lp c suggested that cell to cell pathways dominate for water transport in roots. However, the calculation of Lp r from Lp c showed that at least 26 % of water transport occurs through the apoplast. Roots had different solute permeabilities ( P sr ) and reflection coefficients ( σ sr ) for the solutes used. The σ sr was below unity for the solutes, which have virtually zero permeability for semi-permeable membranes. Conclusions: Suberized endodermis significantly reduces Lp r of seminal roots. The water and solute transport across barley roots is composite in nature and they do not behave like ideal osmometers. The composite transport model should be extended by adding components arranged in series (cortex, endodermis) in addition to the currently included components arranged in parallel (apoplastic, cell to cell pathways).

Transcriptomic changes in barley leaves induced by alcohol ethoxylates indicate potential pathways of surfactant detoxification.

Hardly anything is known regarding the detoxification of surfactants in crop plants, although they are frequently treated with agrochemical formulations. Therefore, we studied transcriptomic changes in barley leaves induced in response to spraying leaf surfaces with two alcohol ethoxylates (AEs). As model surfactants, we selected the monodisperse tetraethylene glycol monododecyl (C12E4) ether and the polydisperse BrijL4. Barley plants were harvested 8 h after spraying with a 0.1% surfactant solution and changes in gene expression were analysed by RNA-sequencing (RNA-Seq). Gene expression was significantly altered in response to both surfactants. With BrijL4 more genes (9724) were differentially expressed compared to C12E4 (6197). Gene families showing pronounced up-regulation were cytochrome P450 enzymes, monooxygenases, ABC-transporters, acetyl- and methyl- transferases, glutathione-S-transferases and glycosyltransferases. These specific changes in gene expression and the postulated function of the corresponding enzymes allowed hypothesizing three potential metabolic pathways of AE detoxification in barley leaves. (i) Up-regulation of P450 cytochrome oxidoreductases suggested a degradation of the lipophilic alkyl residue (dodecyl chain) of the AEs by ω- and ß- oxidation. (ii) Alternatively, the polar PEG-chain of AEs could be degraded. (iii) Instead of surfactant degradation, a further pathway of detoxification could be the sequestration of AEs into the vacuole or the apoplast (cell wall). Thus, our results show that AEs lead to pronounced changes in the expression of genes coding for proteins potentially being involved in the detoxification of surfactants.

The exodermis: A forgotten but promising apoplastic barrier.

The endodermis and exodermis are widely recognized as two important barriers in plant roots that play a role in regulating the movement of water and ions. While the endodermis is present in nearly all plant roots, the exodermis, characterized by Casparian strips and suberin lamellae is absent in certain plant species. The exodermis can be classified into three types: uniform, dimorphic, and inducible exodermis. Apart from its role in water and ion transport, the exodermis acts as a protective barrier against harmful substances present in the external environment. Furthermore, the exodermis is a complex barrier influenced by various environmental factors, and its resistance to water and ions varies depending on the type of exodermis and the maturity of the root. Therefore, investigations concerning the exodermis necessitate a plant-specific approach. However, our current understanding of the exodermal physiological functions and molecular mechanisms governing its development is limited due to the absence of an exodermis in the model plant Arabidopsis. Due to that, unfortunately, the exodermis has been largely overlooked until now. In this review, we aim to summarize the current fundamental knowledge regarding the exodermis in common research used crop species and propose suggestions for future research endeavors.

siRNA-Finder (si-Fi) Software for RNAi-Target Design and Off-Target Prediction.

RNA interference (RNAi) is a technique used for transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS). PTGS is an ubiquitous basic biological phenomenon involved in the regulation of transcript abundance and plants' immune response to viruses. PTGS also mediates genomic stability by silencing of retroelements. RNAi has become an important research tool for studying gene function by strong and selective suppression of target genes. Here, we present si-Fi, a software tool for design optimization of RNAi constructs necessary for specific target gene knock-down. It offers efficiency prediction of RNAi sequences and off-target search, required for the practical application of RNAi. si-Fi is an open-source (CC BY-SA license) desktop software that works in Microsoft Windows environment and can use custom sequence databases in standard FASTA format.

Suberized transport barriers in Arabidopsis, barley and rice roots: From the model plant to crop species.

Water is the most important prerequisite for life and plays a major role during uptake and transport of nutrients. Roots are the plant organs that take up the major part of water, from the surrounding soil. Water uptake is related to the root system architecture, root growth, age and species dependent complex developmental changes in the anatomical structures. The latter is mainly attributed to the deposition of suberized barriers in certain layers of cell walls, such as endo- and exodermis. With respect to water permeability, changes in the suberization of roots are most relevant. Water transport or hydraulic conductivity of roots (Lpr) can be described by the composite transport model and is known to be very variable between plant species and growth conditions and root developmental states. In this review, we summarize how anatomical structures and apoplastic barriers of roots can diversely affect water transport, comparing the model plant Arabidopsis with crop plants, such as barley and rice. Results comparing the suberin amounts and water transport properties indicate that the common assumption that suberin amount negatively correlates with water and solute transport through roots may not always be true. The composition, microstructure and localization of suberin may also have a great impact on the formation of efficient barriers to water and solutes.