Description of a Novel Allelic “Thick Leafed” Mutant of Sorghum

An allelic sorghum [Sorghum bicolor (L.) Moench] mutant with thick and narrow erect leaves (Thl) and reduced adaxial stomatal density was isolated from the Annotated Individually pedigreed Mutagenized Sorghum mutant library developed at the Plant Stress and Germplasm Development Unit at Lubbock TX. The mutant, Thl, was isolated from a pedigreed M3 family generated by ethyl methanesulfonate mutagenization from an elite inbred sorghum line, BTx623, which had been used to sequence the sorghum genome. The mutant has been backcrossed to the wild-type BTx623 confirming that the trait results derive from a stable recessive nuclear gene mutation. Herein, we briefly described morphological and selected physiological characteristics of this mutant sorghum.


Introduction
Herein we described an allelic erect leafed [1] sorghum with thick and narrow leaves developed by methane sulfonate induced mutagenesis [2] [3] of BTx623 [4]. This "thick leaf" (Thl) allelic mutant may help to identify genes influencing 1 The US Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program (Not all prohibited bases apply to all programs.) Persons with disabilities who required alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer. leaf development through mutant mapping, taking advantage of the already sequenced uniform genetic background of BTx623 in which the mutation is embedded [5]. Together with the previously reported leaf architecture mutants [1], this allelic thick leafed mutant may be used to investigate and improve sorghum canopy architecture with the goal of increasing light penetration through the canopy and reducing water use while maintaining or increasing biomass, forage, or grain production. The trait might also be useful in conferring aphid resistance by increasing the distance between the phloem and epidermis.

Mutant Generation and Selection
A sorghum mutant library was generated as previously described [1] [2]. Briefly, air-dried BTx623 seeds were soaked with gentle agitation in concentrations (0.10, 0.15, 0.20, or 0.25%; w/v) of aqueous ethyl methane sulfonate, for 16 h at 25˚C, thoroughly rinsed with distilled water and dried. These seeds were designated as first generation mutant (M1) seeds. These M1 seeds were planted, the plants allowed to develop, and to self pollinate by bagging the panicles with 400-weight rainproof paper pollination bags (Lawson Pollinating Bags 2 , Northfield IL; http://www.lawsonbags.com/) obtained from a distributor (Seedburo Equipment Co., Des Plains IL; http://www.seedburo.com/) after heading and before anthesis to prevent cross-pollination. Panicles setting seeds were manually harvested, individually threshed, planted as M2 plots, allowed to self pollinate with bagging, and a single fertile panicle selected as a source of M3 seeds. Each M3 family of seeds was subsequently planted as a single plot for phenotype evaluation and selection.
The subsequently identified mutant displayed an erect, narrow, thickened leaf in both greenhouse and field. Individual plants within the M3 plots were examined for leaf erectness as described earlier [1]. Leaves that did not droop and maintained erectness as a more acute angle between the leaf and the shoot were identified, but in addition to the erect leafed phenotype, plants with narrower leaves that subjectively felt "thicker" than the wild type (WT) BTx623 were selected. Plants were also grown in plots at the USDA facility (33˚35'38.20" N, 101˚54'11.07"W) and on DOY 180 of the 2017 growing season, Btx-623 and Thl seeds were planted in plots at a depth of 1.5 cm into North-South oriented rows on raised beds spaced 1 m at a rate of 20 seeds/m. The soil at the USDA location is classified as an Amarillo fine sandy loam (fine-loamy, mixed, superactive, thermic Aridic Paleustalfs). After planting, the plots were furrow irrigated several times to induce emergence and ensure an even stand. After the plants reached the fifth to eighth leaf growth stage no more irrigation water was applied. Environmental conditions were recorded by a weather station located 100 m east from the plots (http://www.lbk.ars.usda.gov/WEWC/weather-pswc-data.aspx).

Morphometric Analysis
Leaf thickness was measured with a digital micrometer (Pittsburgh® Tool Item # 47257, Harbor Freight Tools, Calabasas CA) with a claimed accuracy of ± 0.03 mm. To minimize error and to increase reproducibility between measurements that could arise by crushing leaf tissues between the micrometer jaws, the instrument was fitted with two polished zinc disks mounted to the jaws with polyacrylate cement. This distributed the compressive force across the surface of the leaf and prevented crushing soft tissues resulting in more consistent, reproducible, and accurate measurements. It was also thought that this would additionally integrate thickness measurement across a larger sampling area across the surface of the leaf. The second leaf below the flag leaf was selected for leaf thickness measurement at anthesis [6].

Microscopy
Microscopy of selected leaf anatomical features and characteristics of field grown plants was performed on the first leaf immediately below the flag leaf at the boot growth stage. Stomatal density was determined from epidermal impressions produced essentially as described by [7]. Leaves were collected from field grown plants, taken to the laboratory, washed, rinsed, blotted dry, dewaxed with an electronic parts cleaner composed of halogenated solvents, allowed to dry, Micrographs of cross sections were produced similarly except that leaves were sectioned with a razor blade, frozen under liquid nitrogen, and the sections held on the electron microscope's cold stage to keep the samples frozen during imaging.

Gas Exchange
Gas exchange measurements were made with portable photosynthesis systems (Model LI-6400, LiCor Inc., Lincoln NE) fitted with LI-6400-02B LED light sources using mixed LED's delivering both red and blue light to leaves within a 2 cm × 3 cm (6 cm 2 ) sample cuvette. Measurements were made during the grain filling growth stage on the first leaf below the flag leaf during a temporal window beginning 4 hours before solar noon and ending no later than 2 hours after solar noon. Inlet air was passed through a 4-L chamber to buffer rapid changes in [CO 2 ] before entering the system.

Results
Growth and development: Field grown plants at the grain filling growth stage placed against black velvet and photographed in the laboratory are shown in of Thl was about double that of the wild type. Leaf area, leaf mass, stem mass, shoot mass and whole shoot biomass of the mutant was one third that of the wild type in vegetative stages through anthesis, but grain yield was only reduced by about 50%. Seed yield of greenhouse grown Thl plants was 62-g/plant ± 10 g and that of the wild type was 133-g/plant ± 6.5 g/plant (P t  0.001, n = 7).
Scanning electron micrographs of representative leaf surfaces are shown in  That is, similarly sized vasculature appeared to be embedded deeper within the photosynthetically active leaf chlorenchyma. Stomatal distribution as density, i.e., numbers mm −2 and stomatal ratio (upper density/lower density) is shown in Figure 3. The lower leaf stomatal density and upper leaf stomatal densities are "stacked" so that lower, upper, and their contribution to total stomatal densities may be more easily seen (Figure 3(a)). Error bars associated with the measure-   (Figure 3(a)). This resulted in a very slight reduction in mean total leaf stomatal density of only modest statistical significance (P t = 0.10, n = 4 leaves).
The resulting effect upon the stomatal distribution as stomatal ratio is shown in Maximal photosynthetic rates occurred at about the same C i levels (Figure 4(b)).

Discussion
It appears that the differences in gas exchanges between the two isolines might be due to a combination of two anatomical features, the increase in leaf thickness and the reduction in the upper stomatal densities. The decrease in quantum D. C. Gitz III et al. yield seems consistent at least in part, with the associated reduction in the upper stomatal density. However, the reasons for the extremely reduced AC i response remained unclear and must await further work. We propose that increased mesophyll resistance and the lower light levels in the lower regions of the Thl leaves may have led to the apparently reduced AC i response. Leaf internal sub-stomatal [CO 2 ] is derived from the water vapor conductance calculated from transpiration rates and from A net [9]. Lower leaf surfaces that are actively transpiring but not actively assimilating might explain, at least in part, the reduced AC i response of Thl as compared to Btx623. This suggests that the mutant might be less water use efficient than the wild type from which it was derived, though extrapolating results obtained from a 2 × 3 cm leaf cuvette to the agronomic level is fraught with assumption. Again, this was beyond the purview of the current work, which was to briefly describe the "thick leafed" allelic mutant, Thl.
Another factor that might have affected the observed AC i response is the distance through which carbon must travel through the mesophyll before reaching the bundle sheathes [10]. Visual examination of the SEM micrographs Figure   2(e) and Figure 2(f)) reveal considerable increase in the distance between the epidermis and the vasculature. While not of direct relevance to the gas exchange rates, it was also noted that the phloem tissues within the vascular bundles were considerably more removed from the lower epidermis in the Thl plants. It was observed that field grown Thl plants were considerably less affected by sugar cane aphids as compared to the Btx623 plants. This was of particular interest because sugar cane aphids are an emerging problem in sorghum production, and because a clear mechanism for sugar cane aphid resistance can be hypothesized.
An aphid proboscis must be at least as long as the distance from the lower epi-D. C. Gitz III et al. dermis to the phloem bundle to feed. It might simply require considerably less effort for aphids to feed on BTx623 vs. Thl. At this time this remains an observation, but one which could be important, and so it is included herein.
The morphological differences between Thl and BTx623 were more pronounced when plants were field grown or when polyhouse grown under higher light conditions experienced in the months near the summer solstice (not shown). When plants were grown in the winter and early spring, morphological differences between the mutant and the wild type were subtle, though these characteristics were not quantified. The physiological characteristics of the plants grown under these lower light conditions were not examined because we were interested in how the morphological responses would affect the physiology. The assumption here was that the morphological differences of Thl as compared to BTx623 were developmental, possibly photomorpogenic, pleiotropic responses to the environmental cues. If so, the lack of gross morphological differences between the two lines (such as thicker narrower leaves) would be associated with a lack of anatomical differences associated with gas exchange (such as stomatal distribution). However, a systematic approach investigating these hypotheses was not undertaken, primarily because it was beyond the purview of the present work. Moreover, growth under low light levels characteristic of the months outside of the growing season is of limited immediate relevance to the applied nature of such studies. This also points to the importance of agronomically relevant conditions in applied studies evaluating the potential utility of plant traits.
Nevertheless, this mutant might be a useful tool for longer term studies investigating leaf developmental processes in sorghum, perhaps with the goal of manipulating traits that would increase light penetration through the canopy, reduce water use while maintaining or increasing forage biomass and grain production, and conferring resistance to sugar cane aphids.

Conclusion
The Thl mutant sorghum line exhibits altered stomatal distribution via increased abaxialization of stomatal numbers and thicker and narrower leaves. These are associated with altered gas exchange. Increased leaf thickness might be a useful trait in conferring sugar cane aphid resistance.