A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry Lucas Bustaa,b,1,2,3, Elizabeth Schmitza,b,1, Dylan K. Kosmac, James C. Schnableb,d, and Edgar B. Cahoona,b,3 aDepartment of Biochemistry, University of Nebraska–Lincoln, Lincoln, NE 68588; bCenter for Plant Science Innovation, University of Nebraska–Lincoln, Lincoln, NE 68588; cDepartment of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557; and dDepartment of Agronomy and Horticulture, University of Nebraska–Lincoln, Lincoln, NE 68583 Edited by Julian I. Schroeder, University of California San Diego, La Jolla, CA, and approved February 1, 2021 (received for review November 17, 2020) Virtually all land plants are coated in a cuticle, a waxy polyester Though plants deploy diverse mechanisms to protect them- that prevents nonstomatal water loss and is important for heat selves against drought and heat, one of the most widespread and drought tolerance. Here, we describe a likely genetic basis for (found in essentially all land plants), is a hydrophobic, aerial a divergence in cuticular wax chemistry between Sorghum bicolor, surface coating called the cuticle. This structure is composed of a a drought tolerant crop widely cultivated in hot climates, and its fatty acid-derived polyester scaffold called cutin, inside and on close relative Zea mays (maize). Combining chemical analyses, het- top of which accumulates wax, a mixture of hydrophobic com- erologous expression, and comparative genomics, we reveal that: 1) pounds that seal the surface against the movement of water (15). sorghum and maize leaf waxes are similar at the juvenile stage but, While cutin makes major contributions to a cuticle’s biome- after the juvenile-to-adult transition, sorghum leaf waxes are rich in chanical properties and, at least in some species, contributes to triterpenoids that are absent from maize; 2) biosynthesis of the pathogen resistance (16, 17), waxes are primarily responsible for majority of sorghum leaf triterpenoids is mediated by a gene that preventing water from crossing the leaf–atmosphere interface maize and sorghum both inherited from a common ancestor but (18, 19). It is well established that wax mixtures vary between plant that is only functionally maintained in sorghum; and 3) sorghum species, organs, tissues, cell types, and across leaf developmental leaf triterpenoids accumulate in a spatial pattern that was previ- stages (20–23). This suggests wax mixtures are tuned to meet the PLANT BIOLOGY ously shown to strengthen the cuticle and decrease water loss at challenges faced by specific plant surfaces in their immediate high temperatures. These findings uncover the possibility for resur- rection of a cuticular triterpenoid-synthesizing gene in maize that environment. This notion is supported by experiments on trans- could create a more heat-tolerant water barrier on the plant’s leaf genic plants with altered wax amounts and/or wax mixture com- surfaces. They also provide a fundamental understanding of sor- positions: They exhibit altered water barrier properties (18, – ghum leaf waxes that will inform efforts to divert surface carbon 24 27). These studies also demonstrate that plant drought toler- to intracellular storage for bioenergy and bioproduct innovations. ance may be improved through cuticle engineering, thus further cuticular wax | drought tolerance | triterpenoids | juvenile-to-adult Significance transition | sorghum bicolor Virtually all above-ground plant surfaces, such as leaf and stem emand for both food and energy are projected to increase exteriors, are covered in a cuticle: a wax-infused polyester. This Dsubstantially over the coming decades (1). Meeting these needs waxy biocomposite is the largest interface between Earth’s while minimizing negative impacts on our environment, health, biosphere and atmosphere. Its chemical composition is not only and fresh water supply is one of the largest challenges currently highly tuned to mediate nonstomatal water loss, but it also faced by humanity. A component of meeting this challenge is self-assembles to produce superhydrophobic surfaces, protects identifying, improving, and deploying high-yield, low-input, mul- against UV radiation, and contains bioactive compounds that tiusecrops(2).Onecandidateissorghum(Sorghum bicolor), a help resist microbial attack. Developing fundamental knowledge multiuse C4 grass crop. Sorghum is exceptionally tolerant of hot of waxy biocomposites, particularly those on crop species, is a and dry climates, being native to arid regions (3, 4), and the genus prerequisite for an understanding of their structure–function contains considerable natural diversity that offers great potential relationships. Here, we uncover a likely genetic basis for the for the improvement of existing biomass and grain varieties, as presence and absence, respectively, of triterpenoids in the leaf — well as sequenced landraces that are adapted to specific, relatively waxes of sorghum and maize compounds previously associ- extreme environments (5, 6). Sorghum has a relatively small, se- ated with creating heat-tolerant cuticular water barriers. quenced genome (7), its grain is naturally gluten-free (8), and it Author contributions: L.B. and E.S. designed research; L.B., E.S., and D.K.K. performed can be genetically transformed (9, 10). Thus, sorghum is an ex- research; J.C.S. contributed new reagents/analytic tools; L.B., E.S., and J.C.S. analyzed cellent study system for advancing our understanding of the data; and L.B., E.S., D.K.K., J.C.S., and E.B.C. wrote the paper. physiological basis for crop drought tolerance and heat resistance. The authors declare no competing interest. This is particularly true when comparisons are made between This article is a PNAS Direct Submission. sorghum and its close relative maize (Zea mays), one of the most This open access article is distributed under Creative Commons Attribution-NonCommercial- widely grown crops in the world, but also a crop that suffers major NoDerivatives License 4.0 (CC BY-NC-ND). yield losses due to drought. Indeed, investigators in this area have 1L.B. and E.S. contributed equally to this work. identified a variety of physiological mechanisms underlying sor- 2Present address: Department of Chemistry and Biochemistry, University of Minnesota ghum’s positive qualities (11–14). However, there is still a critical Duluth, Duluth, MN 55812. need for mechanistic details underlying these processes and how 3To whom correspondence may be addressed. Email: [email protected] or such differ between sorghum and maize. Until these gaps are fil- [email protected]. led, it will be difficult to further enhance sorghum’s positive This article contains supporting information online at https://www.pnas.org/lookup/suppl/ qualities, and it will not be possible to transfer these qualities to doi:10.1073/pnas.2022982118/-/DCSupplemental. other crop species such as maize. Published March 15, 2021. PNAS 2021 Vol. 118 No. 12 e2022982118 https://doi.org/10.1073/pnas.2022982118 | 1of12 Downloaded by guest on September 29, 2021 underscoring the importance of understanding the waxes of a the common ancestor of maize and sorghum that relies on a drought tolerant plant like sorghum. neofunctionalized steroid biosynthesis gene capable of generating Based on the importance of sorghum waxes, the primary ob- uncommon triterpenoid wax chemicals. Our analyses show that jective of this study was to develop our fundamental knowledge this gene is present in the maize genome but is truncated and not in this area, particularly in a comparative context with maize. expressed. This, together with reports of triterpenoids being ab- Previous work has demonstrated that the juvenile-to-adult tran- sent from the maize leaf surface (30), suggest a likely genetic basis sition, a developmental event in a plant’s life cycle, can be asso- for the observed divergence in sorghum and maize leaf wax ciated with major changes in surface wax characteristics (28, 29). chemistry. Accordingly, this investigation began by analyzing sorghum leaf waxes over the course of the plant’s development, then aimed to Results and Discussion understand underlying biosynthetic processes as well as the evo- Wax Mixtures from Leaves of Adult Sorghum Plants Contain lutionary history of leaf wax biosynthesis in sorghum, all relative to Uncommon Hopane Triterpenoids. The first objective of this study analogous processes in maize. To accomplish this, the present was to identify the chemical constituents on S. bicolor leaf sur- study combined detailed chemical analyses of the sorghum leaf faces. For this, the highest leaf with an exposed ligule on each of surface, bioinformatics-guided characterization of sorghum wax 13 sorghum plants 14 to 98 d of age was collected, pooled, and synthesis genes by heterologous expression, as well as ancestral waxes were extracted. An aliquot of this extract was separated state reconstruction and a comparative genomics analysis of crit- using thin layer chromatography (TLC), resulting in seven prom- ical sorghum wax synthesis genes across six grass species, including inent bands that were each subsequently removed from the plate. sorghum and maize. Considered together, the results indicate that The contents of each band were then analyzed using gas sorghum has maintained a form of wax synthesis likely preset in chromatography-mass spectrometry (GC-MS), resulting in the AB Fig. 1. Leaf wax constituents from S. bicolor.(A)