Cuticular Wax Deposition Research Articles

Metabolite and transcriptome reveal the lipid-associated key components and genes regulated by BoORP3a in ornamental kale.

BoORP3a, an oxysterol-binding protein, located in the endoplasmic reticulum (ER), may function in cuticular wax deposition in ornamental kale. In this study, we investigated its regulation of the key components of cuticular wax and lipids, metabolic pathways, and potential target genes. HS-SPME/GC-MS identified 34 and 31 volatile organic compounds in wild-type and the BoORP3a-overexpressing plant OE-ORP3a-7, respectively, primarily including alkane, ketone, ester, and alcohol. Hentriacontane, 15-nonacosanone, and > C20 alkanes were more abundant in OE-ORP3a-7, which may result in more cuticular wax in this plant. RNA sequencing identified 223 differentially expressed genes (DEGs) between wild-type and OE-ORP3a-7, comprising 119 upregulated and 104 downregulated DEGs. The KEGG enrichment analysis revealed that the downregulated DEGs in OE-ORP3a-7 were involved in glyoxylate and dicarboxylate metabolism, SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) interactions in vesicular transport, fatty acid biosynthesis, and glycerolipid metabolism; the upregulated DEGs were involved in steroid biosynthesis, fatty acid degradation, alpha-linolenic acid metabolism, and sphingolipid metabolism. Bo1g106990, Bo1g123670, and Bo9g166090 were identified as key DEGs in lipid-related pathways. We speculate that BoORP3a regulates several lipid metabolisms and may coordinate lipid turnover and remodeling. The results of this study will enrich the functionality of the ORPs family, provide new insights into plant wax research, and have significant implications for ornamental kale breeding.

PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

Leucine-rich repeat receptor kinase BM41 regulates cuticular wax deposition in sorghum.

Cuticular wax (CW) is the first defensive barrier of plants that forms a waterproof barrier, protects the plant from desiccation, and defends against insects, pathogens, and UV radiation. Sorghum, an important grass crop with high heat and drought tolerance, exhibits a much higher wax load than other grasses and the model plant Arabidopsis. In this study, we explored the regulation of sorghum CW biosynthesis using a bloomless mutant. The CW on leaf sheaths of the bloomless 41 (bm41) mutant showed significantly reduced very long-chain fatty acids (VLCFAs), triterpenoids, alcohols, and other wax components, with an overall 86% decrease in total wax content compared with the wild type. Notably, the 28-carbon and 30-carbon VLCFAs were decreased in the mutants. Using bulk segregant analysis, we identified the causal gene of the bloomless phenotype as a leucine-rich repeat transmembrane protein kinase. Transcriptome analysis of the wild-type and bm41 mutant leaf sheaths revealed BM41 as a positive regulator of lipid biosynthesis and steroid metabolism. BM41 may regulate CW biosynthesis by regulating the expression of the gene encoding 3-ketoacyl-CoA synthase 6. Identification of BM41 as a new regulator of CW biosynthesis provides fundamental knowledge for improving grass crops' heat and drought tolerance by increasing CW.

Wheat MIXTA-like Transcriptional Activators Positively Regulate Cuticular Wax Accumulation.

MIXTA-like transcription factors AtMYB16 and AtMYB106 play important roles in the regulation of cuticular wax accumulation in dicot model plant Arabidopsis thaliana, but there are very few studies on the MIXTA-like transcription factors in monocot plants. Herein, wheat MIXTA-like transcription factors TaMIXTA1 and TaMIXTA2 were characterized as positive regulators of cuticular wax accumulation. The virus-induced gene silencing experiments showed that knock-down of wheat TaMIXTA1 and TaMIXTA2 expressions resulted in the decreased accumulation of leaf cuticular wax, increased leaf water loss rate, and potentiated chlorophyll leaching. Furthermore, three wheat orthologous genes of ECERIFERUM 5 (TaCER5-1A, 1B, and 1D) and their function in cuticular wax deposition were reported. The silencing of TaCER5 by BSMV-VIGS led to reduced loads of leaf cuticular wax and enhanced rates of leaf water loss and chlorophyll leaching, indicating the essential role of the TaCER5 gene in the deposition of wheat cuticular wax. In addition, we demonstrated that TaMIXTA1 and TaMIXTA2 function as transcriptional activators and could directly stimulate the transcription of wax biosynthesis gene TaKCS1 and wax deposition gene TaCER5. The above results strongly support that wheat MIXTA-Like transcriptional activators TaMIXTA1 and TaMIXTA2 positively regulate cuticular wax accumulation via activating TaKCS1 and TaCER5 gene transcription.

A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

Cuticular wax biosynthesis in blueberries (Vaccinium corymbosum L.): Transcript and metabolite changes during ripening and storage affect key fruit quality traits.

In fruits, cuticular waxes affect fruit quality traits such as surface color at harvest and water loss during postharvest storage. This study investigated the transcriptional regulation of cuticular wax deposition in northern highbush blueberries (Vaccinium corymbosum L.) in relation to fruit water loss and surface color during ripening and postharvest storage, as well as the effects of abscisic acid (ABA)-mediated changes in cuticular wax deposition on these fruit quality traits. Total cuticular wax content (μg∙cm-2) decreased during fruit ripening and increased during postharvest storage. Transcriptome analysis revealed a transcript network for cuticular wax deposition in blueberries. Particularly, five OSC-Likes were identified as putative genes for triterpene alcohol production, with OSC-Like1 and OSC-Like2 encoding mixed amyrin synthases, OSC-Like3 encoding a lupeol synthase, and OSC-Like4 and OSC-Like5 encoding cycloartenol synthases. The expression of three CYP716A-like genes correlated to the accumulation of two triterpene acids oleanolic acid and ursolic acid, the major wax compounds in blueberries. Exogenous ABA application induced the expression of triterpenoid biosynthetic genes and the accumulation of β-amyrin and oleanolic acid, as well as increased the ratio of oleanolic acid to ursolic acid. These changes were associated with reduced fruit water loss. The content of β-diketones was also increased by ABA application, and this increase was associated with increased fruit lightness (measured as L* using CIELAB Color Space by a colorimeter). This study provided key insights on the molecular basis of cuticular wax deposition and its implications on fruit quality traits in blueberries.

Genetic variation in ZmWAX2 confers maize resistance to Fusarium verticillioides.

Fusarium verticillioides (F. verticillioides) is a widely distributed phytopathogen that incites multiple destructive diseases in maize, posing a grave threat to corn yields and quality worldwide. However, there are few reports of resistance genes to F. verticillioides. Here, we reveal that a combination of two single nucleotide polymorphisms (SNPs) corresponding to ZmWAX2 gene associates with quantitative resistance variations to F. verticillioides in maize through a genome-wide association study. A lack of ZmWAX2 compromises maize resistance to F. verticillioides-caused seed rot, seedling blight and stalk rot by reducing cuticular wax deposition, while the transgenic plants overexpressing ZmWAX2 show significantly increased immunity to F. verticillioides. A natural occurrence of two 7-bp deletions within the promoter increases ZmWAX2 transcription, thus enhancing maize resistance to F. verticillioides. Upon Fusarium stalk rot, ZmWAX2 greatly promotes the yield and grain quality of maize. Our studies demonstrate that ZmWAX2 confers multiple disease resistances caused by F. verticillioides and can serve as an important gene target for the development of F. verticillioides-resistant maize varieties.

How Does Stomatal Density and Residual Transpiration Contribute to Osmotic Stress Tolerance?

Osmotic stress that is induced by salinity and drought affects plant growth and development, resulting in significant losses to global crop production. Consequently, there is a strong need to develop stress-tolerant crops with a higher water use efficiency through breeding programs. Water use efficiency could be improved by decreasing stomatal transpiration without causing a reduction in CO2 uptake under osmotic stress conditions. The genetic manipulation of stomatal density could be one of the most promising strategies for breeders to achieve this goal. On the other hand, a substantial amount of water loss occurs across the cuticle without any contribution to carbon gain when the stomata are closed and under osmotic stress. The minimization of cuticular (otherwise known as residual) transpiration also determines the fitness and survival capacity of the plant under the conditions of a water deficit. The deposition of cuticular wax on the leaf epidermis acts as a limiting barrier for residual transpiration. However, the causal relationship between the frequency of stomatal density and plant osmotic stress tolerance and the link between residual transpiration and cuticular wax is not always straightforward, with controversial reports available in the literature. In this review, we focus on these controversies and explore the potential physiological and molecular aspects of controlling stomatal and residual transpiration water loss for improving water use efficiency under osmotic stress conditions via a comparative analysis of the performance of domesticated crops and their wild relatives.

β-ketoacyl-CoA synthase improves the drought tolerance of root restricted grown grapevines by regulating the cuticular wax biosynthesis

• Twenty-five VvKCS genes were identified in grapevine ( Vitis vinifera L.). • VvKCS12 and VvKCS14 were induced by root restriction and drought stress. • Overexpression of VvKCS12 and VvKCS14 improved wax biosynthesis in Arabidopsis. • Overexpression of VvKCS12 and VvKCS14 enhanced drought tolerance in Arabidopsis. Drought stress limits fruit ripening and quality of grape cultivar, particularly with the use of root restriction. However, the mechanism involved in the molecular response to water deficit concomitant to agriculture adjustment remains poorly understood. In the present study, the regulation of two grape β-ketoacyl-CoA synthase ( KCS ) genes obtained with transcriptome analysis whose expression is significantly up-regulated under root restriction. Twenty-five KCS coding genes were identified and analyzed from grapevine genome. Two VvKCSs ( VvKCS12 and VvKCS14 ) had one major transcript expressed at a significantly higher level and up-regulated by drought stress in grape leaves. Subcellular localization analysis demonstrated that VvKCS12 and VvKCS14 were located on endoplasmic reticulum (ER). Heterologous overexpression of VvKCS12 and VvKCS14 promoted leaves cuticular wax biosynthesis and deposition, and enhanced drought tolerance in Arabidopsis. These findings underscore the roles of VvKCS12 and VvKCS14 in the drought response of grapes due to root restriction and pave the way for advanced study of other different functions.

Overexpression of ZxABCG11 from Zygophyllum xanthoxylum enhances tolerance to drought and heat in alfalfa by increasing cuticular wax deposition

Drought and heat stresses cause yield losses in alfalfa, a forage crop cultivated worldwide. Improving its drought and heat tolerance is desirable for maintaining alfalfa productivity in hot, arid regions. Cuticular wax forms a protective barrier on aerial surfaces of land plants against environmental stresses. ABCG11 encodes an ATP binding cassette (ABC) transporter that functions in the cuticular wax transport pathway. In this study, ZxABCG11 from the xerophyte Zygophyllum xanthoxylum was introduced into alfalfa by Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (WT), transgenic alfalfa displayed faster growth, higher wax crystal density, and thicker cuticle on leaves under normal condition. Under either drought or heat treatment in greenhouse conditions, the plant height and shoot biomass of transgenic lines were significantly higher than those of the WT. Transgenic alfalfa showed excellent growth and 50% greater hay yield than WT under field conditions in a hot, arid region. Overexpression of ZxABCG11 up-regulated wax-related genes and resulted in more cuticular wax deposition, which contributed to reduction of cuticle permeability and thus increased water retention and photosynthesis capacity of transgenic alfalfa. Thus, overexpression of ZxABCG11 can simultaneously improve biomass yield, drought and heat tolerance in alfalfa by increasing cuticular wax deposition. Our study provides a promising avenue for developing novel forage cultivars suitable for planting in hot, arid, marginal lands.

Co-expression of stress-responsive regulatory genes, MuNAC4, MuWRKY3 and MuMYB96 associated with resistant-traits improves drought adaptation in transgenic groundnut (Arachis hypogaea l.) plants.

Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.

Transcriptome profiling of the chilling response in wheat spikes: II, Response to short-term cold exposure

In this paper we use transcriptome profiling to compare the 4 h and 12 h chilling response in young microspore stage spikes of two wheat lines with contrasting chilling tolerance: chilling-sensitive Wyalkatchem (Wk) and tolerant Young (Yng). In contrast to the previously published paper (4-days chilling), the shorter overnight chilling treatment allowed us to focus on the diversification of early regulatory and metabolic processes in the two wheat lines. The transcriptome results show significant quantitative and qualitative differences in gene expression at the two time points. After 4 h, Wk shows significantly more differentially expressed genes (DEGs) compared to Yng, but a large proportion of these genes are repressed. In Yng, the 4 h cold response is more restrained and most DEGs are cold-induced. After 12 h, the DEG numbers are more comparable between the two lines. GO enrichment analysis was used to identify and compare cold-affected gene functions. After 4 h chilling treatment, genes involved in lipid, sugar, and cell wall metabolism, as well as hormone signalling (SAUR, PP2C, calcium signalling) were mainly repressed in sensitive line Wk, while changes in regulatory factors (APO1, FAR-1, SKP-1) were most notable in tolerant Yng. At both the 4 h and 12 h time points several transcription factors are affected by chilling (Myb, Zinc-finger, bHLH), but the individual genes differ between both wheat lines. After 12 h chilling, LEA genes were induced in Wk only. After 12 h chilling, several SAUR genes are repressed in both wheat lines. Comparison of the response to chilling of lipid metabolism genes indicates that membrane modification starts earlier in tolerant Yng compared to Wk. In contrast, genes involved in deposition of cuticular wax are activated earlier in Wk compared to Yng, but there are significant differences in the response of both wheat lines. Induction of CBF/DREB factors involved in mounting the cold acclimation response occurs earlier in cold-tolerant Yng. The data presented in this paper provide new insights about dynamic and qualitative differences in chilling responses between chilling sensitive and tolerant wheat lines, as well as differences in the kinetics of establishing chilling acclimation.

Microscopic and Transcriptomic Comparison of Powdery Mildew Resistance in the Progenies of Brassica carinata × B. napus.

Powdery mildew is a widespread disease in rapeseed due to a lack of resistant germplasm. We compared the foliar epidermal features and transcriptomic responses between the resistant (R) and susceptible (S) plants among the two parents and progenies of Brassica carinata × B. napus. The amount of cuticular wax and callose deposition on the R plants was much lower than that on the S plants; hence, these chemicals are not all essential to pre-penetration resistance, although the cuticular wax on the R plants had more needle-like crystals. A total of 1049 genes involved in various defense responses were expressed differentially among the R/S plants. The expression levels of two well-known susceptibility genes, MLO6 and MLO12, were much lower in the R plant, indicating an important role in PM resistance. A set of genes related to wax biosynthesis (KCS6, LACS2, CER and MAH1), cell wall modification (PMR5, PMEI9, RWA2, PDCB1 and C/VIF2), chloroplast function (Chlorophyllase-1, OEP161, PSBO1, CP29B and CSP41b), receptor kinase activity (ERECTA, BAK1, BAM2, LYM1, LYM3, RLK902, RLP11, ERL1 and ERL2), IPCS2, GF14 lambda, RPS4 and RPS6 were highly expressed in the R plants. In the S plants, most highly expressed genes were involved in later defense responses, including CERK1, LYK4, LIK1, NIMIN-1, CHITINASE 10, PECTINESTERASE, CYP81F2 and RBOHF and the genes involved in salicylic acid-dependent systemic acquired resistance and hypersensitive responses, indicating the occurrence of severe fungal infection. The results indicate that some uncertain pre-penetration defenses are pivotal for high resistance, while post-penetration defenses are more important for the S plant survival.

Transcriptome profiling of the chilling response in wheat spikes: I, acclimation response to long-term chilling treatment

Chilling conditions coinciding with the early stage of flowering cause significant yield losses in temperate climate zone wheat crops. Progress in breeding for chilling tolerance has been hampered by a lack in understanding of the physiological basis of the problem. Phenotyping methods have therefore used grain yield related traits, rather than traits that focus directly on the cause of the problem. In this study we used transcriptome profiling to gain a better understanding about the differences in cold acclimation in young microspore (YM) stage wheat spikes of a chilling-tolerant (Young, Yng) and a chilling-sensitive (Wyalkatchem, Wk) wheat variety. Gene expression studies were carried out for a longer-term (4-days) controlled environment cyclic chilling treatment (21 °C day/−3 °C night). The accompanying paper addresses the short-term response to chilling using the same treatment cycle (4 h and 12 h), enabling us to compare the regulatory and cold acclimation responses. We used Gene Ontology (GO) enrichment analysis to objectively identify processes that differ in the chilling response of both wheat lines. The transcriptome analysis reveals significant quantitative and qualitative differences between the two wheat lines. Most notable are differences in the expression of genes involved in lipid metabolism and cuticular wax deposition. Several genes involved in auxin metabolism and signalling were enriched in chilling-tolerant Yng compared to Wk, including genes of the Small Auxin Up-Regulated (SAUR) gene family. The results indicate that the chilling tolerant and sensitive wheat varieties differ significantly in their adaption of lipid metabolism and cuticular wax deposition to chilling temperatures. This suggests that controlling membrane fluidity and permeability of the cell wall play a crucial role in controlling chilling tolerance. The results also indicate that auxin metabolism and signalling differentiate the chilling tolerant and sensitive wheat lines, suggesting that this hormone may play an important role in establishing chilling tolerance.

PtoMYB142, a poplar R2R3-MYB transcription factor, contributes to drought tolerance by regulating wax biosynthesis.

Drought is one of the main environmental factors that limit plant development and growth. Accordingly, plants have evolved strategies to prevent water loss under drought stress, such as stomatal closure, maintenance of root water uptake, enhancement of stem water transport, and synthesis and deposition of cuticular wax. However, the molecular evidence of cuticular wax biosynthesis regulation in response to drought is limited in woody plants. Here, we identified an MYB transcription factor, Populus tomentosa Carr. MYB transcription factor (PtoMYB142), in response to drought stress from P. tomentosa. Over-expression of PtoMYB142 (PtoMYB142-OE) resulted in increased wax accumulation in poplar leaves, and significantly enhanced drought resistance. We found that the expression of wax biosynthesis genes CER4 and 3-ketoacyl CoA synthase (KCS) were markedly induced under drought stress, and significantly up-regulated in PtoMYB142-OE lines. Biochemical analysis confirmed that PtoMYB142 could directly bind to the promoter of CER4 and KCS6, and regulate their expression in P. tomentosa. Taken together, this study reveals that PtoMYB142 regulates cuticular wax biosynthesis to adapt to water-deficient conditions.

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.

Inactivation of BoORP3a, an oxysterol-binding protein, causes a low wax phenotype in ornamental kale.

Identifying genes associated with wax deposition may contribute to the genetic improvement of ornamental kale. Here, we characterized a candidate gene for wax contents, BoORP3a, encoding an oxysterol-binding protein. We sequenced the BoORP3a gene and coding sequence from the high-wax line S0835 and the low-wax line F0819, which revealed 12 single nucleotide polymorphisms between the two lines, of which six caused five amino acids substitutions. BoORP3a appeared to be relatively well conserved in Brassicaceae, as determined by a phylogenetic analysis, and localized to the endoplasmic reticulum and the nucleus. To confirm the role of BoORP3a in wax deposition, we generated three orp3a mutants in a high-wax kale background via CRISPR/Cas9-mediated genome editing. Importantly, all three mutants exhibited lower wax contents and glossy leaves. Overall, these data suggest that BoORP3a may participate in cuticular wax deposition in ornamental kale.

Molecular and Physiological Perspectives of Abscisic Acid Mediated Drought Adjustment Strategies.

Drought is the most prevalent unfavorable condition that impairs plant growth and development by altering morphological, physiological, and biochemical functions, thereby impeding plant biomass production. To survive the adverse effects, water limiting condition triggers a sophisticated adjustment mechanism orchestrated mainly by hormones that directly protect plants via the stimulation of several signaling cascades. Predominantly, water deficit signals cause the increase in the level of endogenous ABA, which elicits signaling pathways involving transcription factors that enhance resistance mechanisms to combat drought-stimulated damage in plants. These responses mainly include stomatal closure, seed dormancy, cuticular wax deposition, leaf senescence, and alteration of the shoot and root growth. Unraveling how plants adjust to drought could provide valuable information, and a comprehensive understanding of the resistance mechanisms will help researchers design ways to improve crop performance under water limiting conditions. This review deals with the past and recent updates of ABA-mediated molecular mechanisms that plants can implement to cope with the challenges of drought stress.

The ARRE RING-Type E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis thaliana by Controlling ECERIFERUM1 and ECERIFERUM3 Protein Levels.

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

ZxABCG11 from the xerophyte Zygophyllum xanthoxylum enhances drought tolerance in Arabidopsis thaliana through modulating cuticular wax accumulation

Cuticular waxes, a waterproofing hydrophobic barrier that coats the aerial surfaces of all terrestrial plants, protects plants from various environmental stresses, particularly drought stress. The molecular basis of cuticular wax deposition in xerophytes (e.g. desert plants), including transport mechanisms of waxes to the cell wall, is poorly understood. Here, ZxABCG11 encoding an ATP binding cassette (ABC) transporter was isolated from the xerophyte Zygophyllum xanthoxylum, since we found it was predominantly expressed in young leaves and induced by salt, drought, heat, and combined drought and heat stresses. To ascertain the function of ZxABCG11, we introduced ZxABCG11 into Arabidopsis thaliana and found an increased abundance of cuticular wax on stems and leaves of the transgenic plants, which also exhibited a thickened cuticle and reduced cuticle permeability. Transcript expression analysis revealed that many genes related to wax biosynthesis and transport were up-regulated in these ZxABCG11 transgenic plants. Furthermore, compared with wild type Arabidopsis, ZxABCG11 transgenic plants displayed a higher biomass and survival rate under drought conditions. Taken together, we propose that the expression of ZxABCG11 in Arabidopsis promotes cuticular wax accumulation and thicker cuticle formation, thereby enhancing drought tolerance.