| As the object of this study, Populus euphratica Oliv. (Euphrates poplar or desert poplar) is an ancient plant grew in Mediterranean region since the Tertiary period. It belongs to Salicaceae Populus and so far is a dominant tree species across the arid & semi-arid regions spreading from Midwestern Asia, Northern Africa, to Southern Europe (approximately 30°～50°N). China has the largest growth area & number of P. euphratica in the world, especially in northwestern regions, including Tarim River Basin & Luntai County of Xinjiang Uygur and Ejina Oasis of Alashan League in Inner Mongolia.P. euphratica has a long growth period. Because of the hostile environments, many of them have strange & weird shapes. There is an old saying describing P. euphratica:"It can live for three thousand years, died but not fall for three thousand years, fall but immortal for three thousand years ". In the culture of chinese ethnic minority, it is considered "wonder wood" or "holy trees" and is always closely connected with blood & phoenix as a symbol of death & resurrection.P. euphratica has excellent tolerance to high temperatures, high salinity and drought. It is considered one of the model species for studies of abiotic stress tolerance in trees. It also has heteromorphic leaves, with the lanceolate leaves representative of juvenile-phase non-reproductive growth, and the serrated broad-oval leaves representative of adult-phase reproductive growth.In this study, leaf ecological features; wax amount, chemical composition, ultrastructure, relative expression of genes associated with cuticle lipid metabolis; cutin monomer amount, chemical composition of the heteromorphic leaves of P. euphratica growing in natural riparian habitats differing in available soil moisture are examined to elucidate whether cuticles form differently on these leaf types or in such habitats, and whether there might exist ecologically important interactions between cuticle function, leaf heterophylly and adaptation to water-limited environments,In concrete terms:(1) By comparing the P. euphratica leaves grow in two sample plots differing in available soil moisture, we found that juvenile-growth trees only have lanceolate shaped leaves, while adult trees have a variety of leaf shapes. Leaves of the same shape grew in wetter environment tend to have larger volume, surface area and tend to be thinner & softer than those grew in drier area. With the same soil water condition, lanceolate leaves have larger volume & surface area but shorter petioles than serrated broad-oval leaves. In both sample plots, lanceolate leaves are mainly distributed in the lower tree-canopy, while serrated broad-oval leaves are mainly distributed in the upper tree-canopy. Cuticles of the lanceolate leaves have higher permeability to water than the serrated broad-oval leaves, and when stomata were closed (when at night time or during drought stress), lanceolate leaves lose water more rapidly than serrated broad-oval leaves.(2) We analyzed epicuticular wax crystalline morphology on lanceolate & serrated broad-oval leaves using SEM. The result showed that both lanceolate & serrated broad-oval leaves of P. euphratica have upright projecting flattened crystals on both the adaxial & abaxial leaf surfaces. Wax crystals on surfaces of lanceolate leaves growing at the drier site, displayed dense ordered flattened crystals projecting perpendicular to the surface, while on surfaces of lanceolate leaves at the wetter site exhibited a less densely disordered distributed array of crystals. The adaxial surfaces of serrated broad-oval leaves growing at the wetter site produced plate-like wax structures, whereas adaxial surfaces of serrated broad-oval leaves at the drier site exhibited heavier appearing and more flattened wax deposits. For both lanceolate & broad ovate leaves, abaxial leaf surface waxes were very similar to adaxial surface waxes,except that abaxial wax deposits appeared slightly thicker and more densely distributed than on the adaxial surfaces, especially for the serrated broad-oval leaves in the drier site.(3) By comparing cuticular waxes of lanceolate & serrated broad-oval leaves, we found that serrated broad-oval leaves had over two fold more total wax per area than lanceolate leaves both at the wetter site or drier site. Alkanes,1-alcohols and free fatty acids constituted the most abundant wax classes. Alkanes were more than half of the total wax in serrated broad-oval leaves at both sites. Lanceolate leaves at the drier site had 56.0% more total wax as well as 1.4-fold more alkanes than those at the wetter site. Serrated broad-oval leaves at the drier site had 73.4% more total wax than serrated broad-oval leaves at the wetter site. Among the three main wax classes, it was the alkanes that differed the most between two sites & two leaf types.Among seven individual cutin monomers quantified, the major monomer was 10,16-diOH C16:0 acid, accounting for more than half of total cutin monomers in all samples. Other components identified were 16-OH C16:o acid,C16:o acid,C18:1 dioicacid,18-OHC18:1 acid,C16:0 dioic acid and 18-OH C18:2 acid. Total cutin monomer amounts on serrated broad-oval leaves were 2.1-fold and 0.9-fold higher than amounts observed on lanceolate leaves collected at the wetter & drier sites, respectively. Lanceolate leaves at the drier site had 1.1-fold more total cutin monomers than observed at the wetter site, associated with a 79.8% increase in 10,16-OH C16:0 acids. Lanceolate leaves at the drier site also had higher amounts of 16-OH C16:0 acid, C16:0 acid, C18:1 dioic acid,18-OH C18:1 acid and C16:0 dioic acid. For serrated broad-oval leaves at the driersite,we measured 31.5%more total cutin monomers, associated with 26.5% more 10,16-OH C16:0 acids, and higher 16-OH C16:0 acids, C16:0 acids, C18:1 dioic acids,18-OH C18:1 acids and C16:0 dioic acids than on serrated broad-oval leaves at the wetter site.(4) The relative expression of genes associated with cuticle lipid metabolism in both leaf types and at both sites was assayed by real-time PCR. We observed many differences in gene expression among the 17 tested genes. Serrated broad-oval leaves showed expression of the CER1, CER4, KCS1 and ATT1 transcripts are being over two fold higher in broad-ovate than lanceolate leaves. Transcript analysis showed that most of the tested genes have quite different expression levels in each of the two riparian habitats for both leaf types.In lanceolate leaves, plants growing in the drier sites showed over twofold increase (log2 scale) in transcript abundance of CER1, WBC11, WBC12 and WIN1 than at the wetter site, with the expression of KCS1 & ATT1 being just under two fold higher at the drier site. By comparison, in serrated broad-oval leaves, only CER1, CER26 and MAH1 were at least two fold more highly expressed in the drier than wetter site. CER1, WBC11, WBC12 and WIN1 transcripts were the most highly expressed in both lanceolate & serrated broad-oval leaves at the drier site.This is the first report describing the cuticle lipids on P. euphratica, a trait we demonstrate is closely associated with its adaptation to arid climates. Significant differences in the cuticular lipids and permeability of heteromorphic leaves are described, with the adult-phase serrated broad-oval leaves exhibiting more abundant cuticle lipids and lower cuticle permeability than juvenile-phase lanceolate leaves, revealing a potential development-associated adaptation for survival in arid environments. Dramatic differences were observed for the cuticle lipids and permeability between the wetter site having much higher soil water availability and the drier site having less available soil water, with the drier site producing leaves with much heavier cuticles (more cuticle waxes & cutin monomers) and lower cuticle permeability. P. euphratica growing in the drier site thus produced a leaf cuticle that is associated with better adaptation to a more xeric environment. CER1 and other cuticle-associated genes were expressed at higher levels in the serrated broad-oval leaves than lanceolate leaves, and at higher levels in the drier site than wetter site, which corresponded with more abundant leaf cuticle lipids and reduced cuticle permeability. These results thus indicate a role for cuticle-associated genes in P. euphratica in adaptation to limited water environments. Further studies are needed to elucidate the function of cuticle lipids, permeability and associated genes in P. euphratica, and their role in providing an adaptive response enhancing growth & survival in arid environments, including the impact of developmental variation in these traits. Further studies are also needed to elucidate the degree to which genome sequence differences may impact the cuticle variation we observed at the two sites.Work presented here provides a new understanding of heterophyllous leaf cuticles in P. euphratica, and lays groundwork for selection of improved P. euphratica genotypes for human use, and the development of new strategies for protecting natural populations of P. euphratica and their associated ecosystems. |
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