Eliana Gonzales-Vigil

Cuticular waxes are a mixture of hydrophobic components protecting plant tissues from the environment. Populus trichocarpa and P. balsamifera are closely-related tree species with broad North American distribution; however, variation in their cuticular wax composition across their range remained poorly understood. To address this gap, stem and leaf waxes from both species across three developmental stages were profiled by gas chromatography-mass spectrometry. A core set of compounds shared across tissues, accessions, and developmental stages was detected, as well as tissue- or species-specific constituents such as phenolics and alkenes. Phenolic-derived compounds were more prevalent at early stages of development compared to the predominantly aliphatic composition of mature tissues. Moreover, early stage leaves also showed enhanced protection from desiccation and oxidative stress in a subset of accessions. Alkenes, found exclusively in leaves, segregated the poplar accessions into alkene-producing and alkene-lacking phenotypes. GWAS analysis of 174 P . trichocarpa accessions identified a region on chromosome 10 containing a tandem cluster of ketoacyl CoA synthases associated with alkene accumulation in P. trichocarpa , which was not significantly associated in 133  P. balsamifera individuals examined. These findings reveal dynamic, developmentally regulated, and species-specific variation in poplar wax profiles. Understanding these chemical traits offers new opportunities to develop Populus varieties with enhanced environmental resilience and adaptive potential.

Cuticular waxes coating leaf surfaces can help plants tolerate drought events by reducing non-stomatal water loss. Despite their role in drought tolerance, little is known about how cuticular wax composition has changed during breeding in Canadian bread wheat (Triticum aestivum L.) varieties. To fill in this gap, flag leaves of the Canadian Heritage Bread Wheat Panel, which include 30 varieties released between 1842 and 2018, were surveyed to determine if and how cuticular wax composition in wheat has changed at two breeding ecozones over this period. Following this, a subset of varieties was subjected to drought conditions to compare their responses. As expected, modern varieties outperformed old varieties with a significantly larger head length and reaching maturity earlier. Yet, when challenged with drought, old varieties were able to significantly increase the accumulation of β-diketones to a higher extent than modern varieties. Furthermore, RNAseq was performed on the flag leaf of four modern varieties to identify potential markers that could be used for selection of higher accumulation of cuticular waxes. This analysis revealed that the W1 locus is a good candidate for selecting higher accumulation of β-diketones. These findings indicate that the variation in cuticular waxes upon drought could be further incorporated in breeding of future bread wheat varieties.

The cuticle is a hydrophobic structure that seals plant aerial surfaces from the surrounding environment. To better understand how cuticular wax composition changes over development, we conducted an untargeted screen of leaf surface lipids from black cottonwood ( Populus trichocarpa ). We observed major shifts to the lipid profile across development, from a phenolic and terpene-dominated profile in young leaves to an aliphatic wax-dominated profile in mature leaves. Contrary to the general pattern, levels of aliphatic cis -9-alkenes decreased in older leaves following their accumulation. A thorough examination revealed that the decrease in cis -9-alkenes was accompanied by a concomitant increase in aldehydes, one of them being the volatile compound nonanal. By applying exogenous alkenes to P. trichocarpa leaves, we show that unsaturated waxes in the cuticle undergo spontaneous oxidative cleavage to generate aldehydes and that this process occurs similarly in other alkene-accumulating systems such as balsam poplar ( Populus balsamifera ) leaves and corn ( Zea mays ) silk. Moreover, we show that the production of cuticular wax–derived compounds can be extended to other wax components. In bread wheat ( Triticum aestivum ), 9-hydroxy-14,16-hentriacontanedione likely decomposes to generate 2-heptadecanone and 7-octyloxepan-2-one (a caprolactone). These findings highlight an unusual route to the production of plant volatiles that are structurally encoded within cuticular wax precursors. These processes could play a role in modulating ecological interactions and open the possibility for engineering bioactive volatile compounds into plant waxes.

Plants protect their tissues from insect herbivory with specialized structures and chemicals, such as cuticles, trichomes, and metabolites contained therein. Bacteria inside the insect gut are also exposed to plant defences and can potentially modify the outcome of plant–insect interactions. To disentangle this complex multi-organism system, we used tomato mutants impaired in the production of plant defences (odorless-2 and jasmonic acid–insensitive1) and two cultivars (Ailsa Craig and Castlemart), exposed them to herbivory by the cabbage looper (Trichoplusia ni H.) and collected the insect frass for bacterial community analysis. While the epicuticular wax and terpene profiles were variable, the leaf fatty acid composition remained consistent among genotypes. Moreover, larval weight confirmed the negative association between plant defences and insect performance. The distinctive frass fatty acid profiles indicated that plant genotype also influences the lipid digestive metabolism of insects. Additionally, comparisons of leaf and insect-gut bacterial communities revealed a limited overlap in bacterial species between the two sample types. Insect bacterial community abundance and diversity were notably reduced in insects fed on the mutants, with Enterobacteriaceae being the predominant group, whereas putatively pathogenic taxa were found in wildtype genotypes. Altogether, these results indicate that plant defences can modulate insect-associated bacterial community composition.

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1–7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in β-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

Sucrose synthase (SuSy) is one of two enzyme families capable of catalyzing the first degradative step in sucrose utilization. Several earlier studies examining SuSy mutants in Arabidopsis failed to identify obvious phenotypic abnormalities compared with wild-type plants in normal growth environments, and as such a functional role for SuSy in the previously proposed cellulose biosynthetic process remains unclear. Our study systematically evaluated the precise subcellular localization of all six isoforms of Arabidopsis SuSy via live-cell imaging. We showed that yellow fluorescent protein (YFP)-labeled SuSy1 and SuSy4 were expressed exclusively in phloem companion cells, and the sus1/sus4 double mutant accumulated sucrose under hypoxic conditions. SuSy5 and SuSy6 were found to be parietally localized in sieve elements and restricted only to the cytoplasm. SuSy2 was present in the endosperm and embryo of developing seeds, and SuSy3 was localized to the embryo and leaf stomata. No single isoform of SuSy was detected in developing xylem tissue of elongating stem, the primary site of cellulose deposition in plants. SuSy1 and SuSy4 were also undetectable in the protoxylem tracheary elements, which were induced by the vascular-related transcription factor VND7 during secondary cell wall formation. These findings implicate SuSy in the biological events related to sucrose translocation in phloem.

Significance: Cellulose, the most abundant biopolymer on earth, is the major constituent of plant cell walls and is ubiquitously used by industry. This biopolymer is made by plasma membrane-localized CELLULOSE SYNTHASE (CESA) enzymes. To transit from deposition of a growing primary cell wall to a strong secondary cell wall, xylem cells must remodel the CESA machinery to express a new set of CESA isoforms specific to secondary cell wall synthesis. We outline a detailed framework for how this change in cellulose synthesis occurs. Our work provides the principles for how plants change their capacity to produce cellulose and therefore plant biomass.

A ketoacyl CoA synthase (PotriKCS1) is key to the biosynthesis of alkenes in the cuticular waxes of poplar leaves.