The role of ABC transporters in cuticular lipid secretion

The aerial surfaces of plants are enveloped by a waxy cuticle, which among other functions serves as a barrier to limit non-stomatal water loss and defend against pathogens. The cuticle is a complex three-dimensional structure composed of cutin (a lipid polyester matrix) and waxes (very long chain fatty acid derivatives), which are embedded within and layered on top of the cutin matrix. Biosynthesis of cuticular lipids is believed to take place solely within aerial epidermal cells. Once synthesized, both the waxes and the cutin precursors must leave the cytoplasm, pass through the hydrophilic apoplastic space, and finally assemble to form the cuticle. These processes of secretion and assembly are essentially unknown. Initial steps toward our understanding of these processes were the characterization of CER5/ABCG12/WBC12 and more recently ABCG11/WBC11, a pair of ABC transporters required for cuticular lipid secretion. ABCG12 is involved in wax secretion, as mutations in this gene result in a lower surface-load of wax and a concomitant accumulation of lipidic inclusions within the epidermal cell cytoplasm. Mutations in ABCG11 result in a similar wax phenotype as cer5 and similar cytoplasmic inclusions. In contrast to cer5, however, abcg11 mutants also show significantly reduced cutin, post-genital organ fusions, and reduced growth and fertility. Thus, for the first time, a transporter is implicated in cutin accumulation. This review will discuss the secretion of cuticular lipids, focusing on ABCG12, ABCG11 and the potential involvement of other ABC transporters in the ABCG subfamily.     

钼在桃树干旱胁迫响应中的作用解析

以毛桃(Amygdalus persica)实生苗为试材, 研究干旱胁迫下, 钼酸铵处理对钼辅因子硫化酶编码基因(LOS5/ABA3)表达量、脱落酸(ABA)含量及抗旱相关生理指标的影响。结果表明, 干旱胁迫下, 喷施不同浓度钼酸铵处理毛桃实生苗叶片, 其含水量及叶绿素和脯氨酸含量显著高于对照, 且以0.04%钼酸铵处理效果最好; 电解质渗漏率显著低于对照。干旱胁迫下, 与对照相比, 喷施0.04%钼酸铵的毛桃实生苗叶片中LOS5/ABA3表达量显著提高; ABA含量、水分利用效率和净光合速率均高于对照, 蒸腾速率低于对照, 且差异显著; 叶片抗氧化酶活性显著升高, MDA含量显著降低; 离体处理的叶片质量损失减缓, 且差异显著。研究表明毛桃实生苗在干旱胁迫下喷施钼酸铵可通过上调钼辅因子硫化酶编码基因的表达水平, 提高叶片中ABA和脯氨酸含量及抗氧化酶活性, 从而缓解干旱胁迫下的细胞膜氧化伤害, 降低叶片失水速率, 减轻干旱胁迫对毛桃实生苗的伤害。     

空气湿度与土壤水分胁迫对紫花苜蓿叶表皮蜡质特性的影响

选用2个抗旱性不同的紫花苜蓿品种,敖汉(强抗旱)和三得利(弱抗旱),设置空气湿度(45%-55%和75%-85%)和土壤水分胁迫(75%和35%田间持水量)处理,分析紫花苜蓿叶表皮蜡质含量、组分及晶体结构、气体交换参数、水势及脯氨酸含量的变化规律。结果表明,单独土壤水分胁迫时,紫花苜蓿叶表皮蜡质晶体结构及蜡质总量无显著变化;敖汉蜡质组分中烷类、酯类含量增加,醇类含量下降;三得利醇类含量下降,烷类、酯类含量变化不显著。低空气湿度胁迫时,两品种蜡质总量无显著变化,烷类和酯类含量显著增加,醇类含量显著下降,叶表皮片状蜡质晶体结构熔融呈弥漫性,扩大了对叶表面积的覆盖,其蒸腾速率显著低于正常湿度。复合胁迫处理时,叶表皮片状蜡质晶体结构继续呈弥漫性,烷类、酯类、未知蜡质组分含量均高于单独胁迫处理,醇类含量最低,而蜡质总量除三得利显著高于对照外,其余均无显著差异。紫花苜蓿叶表皮蜡质各组分含量(除醇类)及蜡质总量与光合速率呈显著负相关,与蒸腾速率无显著相关关系。蜡质总量与叶水势呈显著正相关。总体上,敖汉蜡质总量显著高于三得利,蜡质组分中烷类物质的增加有助于提高植株的抗旱性。在复合胁迫下,强抗旱品种主要通过气孔因素控制水分散失,而弱抗旱品种通过气孔和非气孔因素共同控制植物水分散失。     

Arabidopsis U‐box E3 ubiquitin ligase PUB11 negatively regulates drought tolerance by degrading the receptor‐like protein kinases LRR1 and KIN7

Both plant receptor‐like protein kinases (RLKs) and ubiquitin‐mediated proteolysis play crucial roles in plant responses to drought stress. However, the mechanism by which E3 ubiquitin ligases modulate RLKs is poorly understood. In this study, we showed that Arabidopsis PLANT U‐BOX PROTEIN 11 (PUB11), an E3 ubiquitin ligase, negatively regulates abscisic acid (ABA)‐mediated drought responses. PUB11 interacts with and ubiquitinates two receptor‐like protein kinases, LEUCINE RICH REPEAT PROTEIN 1 (LRR1) and KINASE 7 (KIN7), and mediates their degradation during plant responses to drought stress in vitro and in vivo. pub11 mutants were more tolerant, whereas lrr1 and kin7 mutants were more sensitive, to drought stress than the wild type. Genetic analyses show that the pub11 lrr1 kin7 triple mutant exhibited similar drought sensitivity as the lrr1 kin7 double mutant, placing PUB11 upstream of the two RLKs. Abscisic acid and drought treatment promoted the accumulation of PUB11, which likely accelerates LRR1 and KIN7 degradation. Together, our results reveal that PUB11 negatively regulates plant responses to drought stress by destabilizing the LRR1 and KIN7 RLKs.     

Characterization of <Emphasis Type="Italic">Glossy1</Emphasis>-homologous genes in rice involved in leaf wax accumulation and drought resistance

The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. Glossy 1 (GL1) is one of the reported genes controlling wax synthesis. This study analyzed GL1-homologous genes in Oryza sativa and characterized the key members of this family involved in wax synthesis and stress resistance. Sequence analysis revealed 11 homologous genes of GL1 in rice, designated OsGL1-1 to  OsGL1-11. OsGL1-1, -2 and -3 are closely related to GL1. OsGL1-4, -5, -6, and -7 are closely related to Arabidopsis CER1 that is involved in cuticular wax biosynthesis. OsGL1-8, -9, -10 and -11 are closely related to SUR2 encoding a putative sterol desaturase also involved in epicuticular wax biosynthesis. These genes showed variable expression levels in different tissues and organs of rice, and most of them were induced by abiotic stresses. Compared to the wild type, the OsGL1-2-over-expression rice exhibited more wax crystallization and a thicker epicuticular layer; while the mutant of this gene showed less wax crystallization and a thinner cuticular layer. Chlorophyll leaching experiment suggested that the cuticular permeability was decreased and increased in the over-expression lines and the mutant, respectively. Quantification analysis of wax composition by GC–MS revealed a significant reduction of total cuticular wax in the mutant and increase of total cuticular wax in the over-expression plants. Compared to the over-expression and wild type plants, the osgl1-2 mutant was more sensitive to drought stress at reproductive stage, suggesting an important role of this gene in drought resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Co-regulation of indole glucosinolates and camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6 signaling pathways

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

一个玉米旱敏感突变体的遗传分析与基因定位

玉米干旱胁迫相关突变体在发掘玉米耐旱关键基因研究中具有重要利用价值。在玉米自交系综31的田间扩繁过程中,发现一个玉米干旱胁迫敏感的自然突变体,该突变体在轻度干旱条件下叶片发生卷曲,严重干旱时叶尖变黄,衰老坏死。遗传分析表明突变性状受1对主效单基因控制,表现为隐性遗传,将突变基因命名为DS。利用B73与突变体ds组配F2分离群体,以干旱条件下叶片是否卷曲为指标,将DS基因初定位在第3号染色体SSR标记umc1772和umc2158之间,物理距离为5 Mb。以上研究结果为该基因的克隆及功能分析奠定了基础。     

Leaf alkanes in Persea and related taxa

The normal-alkane range in leaf cuticular waxes from 22 Persea species and cultivars and from the closely related genus Beilschmiedia was C₂₃ H₄₈ to C₃₅, H₇₂; alkanes with an odd number of carbon atoms predominated, C₃₃ H₆₈ usually constituting about half of the total wax. The alkane profiles gave good agreement with established taxonomy. Beilschmiedia showed an alkane distribution quite different from that of the Persea taxa. Amongst Persea species, the geographical and phylogenetic distinctiveness of P. indica and P. donnell-smithii were reflected in the distinctiveness of their alkanes. Within the subgenus Persea. the morphologically most distinct entity. P. schiedeana, also had a distinct alkane profile. Cultivars of hybrid origin indicated in their alkane proportions pronounced gene interaction.     

Cloning of the <Emphasis Type="Italic">Brcer1</Emphasis> gene involved in cuticular wax production in a glossy mutant of non-heading Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. var. <Emphasis Type="Italic">communis</Emphasis>)

Cuticular wax is a complex mixture of very-long-chain fatty acid derivatives. The wax on the surface of plants serves as a protective barrier to reduce non-stomatal water loss and environmental damage. However, the loss of wax may lead to a glossy phenotype, which is an favorable trait in leafy vegetables. The mechanism of glossy mutants in non-heading Chinese cabbage (Brassica rapa L. var. communis) has not been studied yet. In this study, scanning electron microscopy (SEM) showed that the cuticular wax on the leaves and stem of a glossy mutant was dramatically reduced compared with that of the wild-type plant. Transmission electron microscopy (TEM) revealed that the cuticle ultrastructure of glossy mutant leaf and stem were altered when compared with the wild type. A cuticle wax analysis showed the total wax content of leaves, as well as alkanes, ketones and alcohols, was decreased. A genetic analysis indicated that the glossy phenotype was controlled by a single gene. Based on a homology analysis, the Brcer1 gene was identified as the candidate gene controlling the glossy phenotype. In the glossy mutant, a 39-bp deletion leads to an mRNA disruption and reduces the expression of the BrCER1 gene. Sequence analysis showed that a loss of function mutation in the Brcer1 gene was different from that of Cgl1, which was previously shown to be responsible for the glossy phenotype in B. oleracea, showing typical parallel selection. These findings provide a better understanding of the cuticular wax biosynthesis pathway and offer important information for molecular-assisted breeding of non-heading Chinese cabbage (B. rapa L. var. communis).     

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.     

Epicuticular Wax and Cuticular Resistance in Rice

High leaf cuticular resistance has been reported as a component adaptation of plants to drought prone regions, Experiments were conducted to evaluate and characterize the role of epicuticular wax as a component of cuticular resistance to water vapor loss from rice (Oryza sativa L.) leaves. This information is necessary to determine the applicability of including higher cuticular resistance in an upland rice breeding program and to evaluate potential selection methods. Diffusion porometry, electron microscopy, and gas liquid chromatography were employed. Measurement of cuticular resistance by leaf diffusive resistance porometry after stomatal closure by exposure of rice leaves to pure CO₂ for 15 min was found sufficient to induce complete stomatal closure regardless of light level, and was superior to dark acclimation for this purpose. Removal of epicuticular wax from rice leaves by chloroform dip significantly reduced the cuticular resistance. Stressed plants were observed to increase cuticular resistance, illustrating the responsive nature of this characteristic. Gas liquid chromatography (GLC) of the chloroform leaf dip proved to be an expedient method of characterizing both quantitative and qualitative differences in the epicuticular wax of rice cullivars. The porometry and GLC techniques may be useful in selecting parents, spot checking in a pedigree program, or checking lines reaching the yield testing stage, but ate not well suited lor mass screening early generation progeny. Significant differences were found in the two rice cultivurs, ‘63–83′, an upland adapted rice from West Africa, and “IR20′, bred and selected in submerged paddy culture in ihc Philippines, by tlie above methods of characterizing cuticular resistance and epicuticular wax. These results are of ecological significance to plant breeders.