Cuticular Wax Composition is Essential for Plant Recovery Following Drought with Little Effect under Optimal Conditions

Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. To answer this fundamental question, we employed genome editing in tree tobacco (Nicotiana glauca) plants and generated mutations in 16 different cuticular lipids-related genes. We chose tree tobacco due to the abundant, yet simply composed epicuticular waxes deposited on its surface. Five out of 9 different mutants that displayed a cuticular lipids-related phenotype were selected for in depth analysis. They had either reduced total wax load or complete deficiency in certain wax components. This led to substantial modification in surface wax crystal structure and to elevated cuticular water loss. Remarkably, under non-stressed conditions, mutant plants with altered wax composition did not display elevated transpiration or reduced growth. However, once exposed to drought, plants lacking alkanes were not able to strongly reduce their transpiration, leading to leaf death and impaired recovery upon resuscitation, and even to stem cracking, a phenomenon typically found in trees experiencing drought stress. In contrast, plants deficient in fatty alcohols exhibited an opposite response, having reduced cuticular water loss and rapid recovery following drought. This deferential response was part of a larger trend, of no common phenotype connecting plants with a glossy appearance. We conclude that alkanes are essential under drought response and much less under normal non-stressed conditions, enabling plants to seal their cuticle upon stomatal closure, reducing leaf death and facilitating a speedy recovery.

Pour point depression and flow improvement of waxy crude oil using polyethylene glycol esters of cashew nut shell liquid

Wax crystallization and deposition is a major flow assurance problem in production and transportation of waxy crude oil. Conventional flow improvers are mainly high molecular weight synthetic polymers, many of which are eco-toxic. Bio-based flow improvers derived from natural products are promising as inexpensive, eco-friendly alternatives to existing products. In this study, natural cashew nut shell liquid (CNSL) extracted from waste shells of Anacardium occidentale was esterified with polyethylene glycol (PEG). CNSL derivative reduced the pour point of waxy crude oil by 12 °C at 1000 ppm. The effects of CNSL derivatives on wax crystal morphology and micro-structure were studied by cross-polarized microscopy. Micrographs were processed and analyzed with ImageJ software. Addition of CNSL derivatives to oil resulted in changes in wax crystal morphology and micro-structure evidenced by a reduction in average crystal Feret diameter and aspect ratio and increase in boundary fractal dimension, indicative of formation of increasing number of smaller, rounder crystals. Effect of the additives on flow properties of the waxy oil was determined using a co-axial cylinder rotational viscometer. Dynamic viscosity of oil at shear rate of 17 s−1 was reduced by 79.7–90.5%. CNSL-PEG esters show good prospects as low-cost additives for production, storage and pipeline transportation of waxy crude oil.

A Novel Method for Characterizing the Aggregation of Wax Crystals and An Improvement in Wax Deposition Modeling

The aggregation behavior and the subsequent deposition behavior of wax crystals own undesirable effects on the production and transportation of waxy crude oil. The understanding and prediction of these behaviors are essential to ensure economic and uninterrupted flow of waxy crude oil when the oil temperature decreases below the wax appearance temperature (WAT). In this paper, a novel method of fractal dimensional analysis was introduced to elucidate the aggregation behavior of wax crystals in different shear flow fields. The fractal methodology for characterizing wax crystal aggregation was then developed, and a blanket algorithm was introduced to compute the fractal dimension of the aggregated wax crystals. Considering the flow characteristics of waxy crude oil in a pipeline can be correlated with the shearing stress work, a modified wax deposition model focusing on shearing energy analysis was established. The results indicate that a quantitative interpretation of the wax crystal aggregation behavior can be realized using the fractal methodology. The aggregation behavior of the wax crystals is closely related to the temperature and shearing experienced by the waxy crude oil. The aggregation behavior will be intensified with decreasing temperature and shearing effect, and a wider fractal dimension distribution appears at lower temperatures when the same shear rate range is used. Furthermore, the improved model provides a method for discussing the effects of the operating conditions on wax deposition. The average relative deviation between the improved model prediction results and experimental results from the literature is 3.01–5.32%.

Ecophysiology with barley eceriferum (cer) mutants: the effects of humidity and wax crystal structure on yield and vegetative parameters

Background and Aims In addition to preventing water loss, plant cuticles must also regulate nutrient loss via leaching. The eceriferum mutants in Hordeum vulgare (barley) potentially influence these functions by altering epicuticular wax structure and composition. Methods Cultivar ‘Bonus’ and five of its cer mutants were grown under optimal conditions for vegetative growth and maturation, and nine traits were measured. Nutrient and water amounts going through the soil and the amount of simulated rain as deionized water, affecting phyllosphere humidity, delivered during either the vegetative or maturation phase, were varied. Cer leaf genes and three wilty (wlt) mutations were characterized for reaction to toluidine blue and the rate of non-stomatal water loss. Key Results Vegetative phase rain on ‘Bonus’ significantly decreased kernel weight and numbers by 15–30 %, while in cer.j59 and .c36 decreases of up to 42 % occurred. Maturation phase findings corroborated those from the vegetative phase. Significant pleiotropic effects were identified: cer.j59 decreased culm and spike length and 1000-kernel weight, .c36 decreased kernel number and weight, .i16 decreased spike length and .e8 increased culm height. Excepting Cer.zv and .ym mutations, none of the other 27 Cer leaf genes or wlt mutations played significant roles, if any, in preventing water loss. Cer.zv and .ym mutants lost non-stomatal water 13.5 times faster than those of Cer.j, .yi, .ys and .zp and 18.3 times faster than those of four cultivars and the mutants tested here. Conclusions Using yield to measure the net effect of phyllosphere humidity and wax crystal structure revealed that the former is far more important than the latter. The amenable experimental setup described here can be used to delve deeper. Significant pleiotropic effects were identified for mutations in four Cer genes, of which one is known to participate in wax biosynthesis. Twenty-seven Cer leaf genes and three wlt mutations have little if any effect on water loss.

Effects of palmitic acid (16:0), hexacosanoic acid (26:0), ethephon and methyl jasmonate on the cuticular wax composition, structure and expression of key gene in the fruits of three pear cultivars

The chemical composition, crystal morphology and expression levels of associated genes involved in the cuticular wax of three pear cultivars ‘Housui’, ‘Cuiguan’ and ‘Yuluxiang’ after treatment with palmitic acid (PA), hexacosanoic acid (HA), ethephon and methyl jasmonate (Meja) were determined. A total of 59 cuticular wax compounds were detected across all samples. The wax coverage of ‘Housui’ fruits increased by 71.74, 93.48 and 89.13% after treatment with PA, ethephon and Meja, respectively, and treatment with PA, HA and Meja also increased the wax coverage in ‘Cuiguan’ (65.33, 20.00 and 21.33% respectively) and in ‘Yuluxiang’ (38.60, 63.16 and 42.11% respectively) fruits. Heatmap clustering analysis and partial least-squares-discriminate analysis (PLS-DA) also revealed that the different treatments exerted various influences on cuticular wax among the different cultivars. In addition, the wax component coverage and wax crystal structures showed variations among the different cultivars as well as different treatments. Gene expression analysis revealed 11 genes likely to be involved in pear fruit wax synthesis, transport and regulation. Taken together, the results of this study demonstrate that the differences in the cuticular waxes of the fruits of different cultivars after treatment with PA, HA, ethephon or Meja might lead to a better understanding of the regulatory effect of a substrate or elicitor on the composition and deposition of cuticular waxes.

Chemical and Transcriptomic Analysis of Cuticle Lipids under Cold Stress in Thellungiella salsuginea

Plant cuticle lipids form outer protective layers to resist environmental stresses; however, the relationship between cuticle properties and cold tolerance is unclear. Here, the extremophyte Thellungiella salsuginea was stressed under cold conditions (4 °C) and the cuticle of rosette leaves was examined in terms of epicuticular wax crystal morphology, chemical composition, and cuticle-associated gene expression. The results show that cold induced formation of distinct lamellas within the cuticle ultrastructure. Cold stress caused 14.58% and 12.04% increases in the amount of total waxes and cutin monomer per unit of leaf area, respectively, probably associated with the increase in total fatty acids. The transcriptomic analysis was performed on rosette leaves of Thellungiella exposed to cold for 24 h. We analyzed the expression of 72 genes putatively involved in cuticle lipid metabolism, some of which were validated by qRT-PCR (quantitative reverse transcription PCR) after both 24 h and one week of cold exposure. Most cuticle-associated genes exhibited higher expression levels under cold conditions, and some key genes increased more dramatically over the one week than after just 24 h, which could be associated with increased amounts of some cuticle components. These results demonstrate that the cuticle provides some aspects of cold adaptation in T. salsuginea.