Reducing stomatal density by expression of a synthetic EPF increases leaf intrinsic water use efficiency and reduces plant water use in a C4crop

38 Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience 39 and expanding cultivation on marginal lands. Reducing stomatal conductance ( gs) 40 through manipulating stomatal density has been observed to translate to improved 41 WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces 42 photosynthetic carbon gain. A different response is expected in C4 plants because 43 they possess specialized anatomy and biochemistry which concentrates CO2 at the 44 site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 45 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are 46 unlikely to impair AN. To test this hypothesis, genetic strategies were investigated to 47 reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic 48 epidermal patterning factor (EPF) transgenic allele in sorghum, lead to reduced 49 stomatal densities. A moderate reduction in stomatal density did not strengthen 50 stomatal limitation to A N, improved WUE, reduced water use, and avoided loss of 51 carbon fixation during a period of water deprivation. However, these positive outcomes 52 were associated with negative pleiotropic effects on reproductive development and 53 photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene 54 to specific tissues provides a potential pathway to optimal agronomic outcomes. 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint

72 Water availability is a critical factor limiting crop productivity worldwide (Boyer 73 1982). Water Use Efficiency (WUE) has been recognized as an important trait for crop 74 improvement for over a century (Briggs and Shantz 1917) because it describes how 75 much productivity can be achieved per unit of water used by the crop. WUE is key to 76 terrestrial plant function because it reflects the inevitable trade -off between losing 77 water vapor from leaves to the atmosphere , while stomata are open , to allow 78 photosynthetic CO 2 uptake. Climate change is impacting precipitation patterns and 79 vapour pressure deficit, which in turn impact water availability and demand in cropping 80 systems (Sadok et al. 2021, Chiang et al. 2021). In silico modelling that incorporates 81 historical yields and projected environmental conditions suggests that drought events 82 will continue to be a key driver of yield losses in the future (Webber et al. 2018). Water 83 supplies for irrigation are limited and unsustainable (WWAP 2015). Demand is 84 increasing for agricultural products from marginal lands (Gelfand et al. 2013; Khanna 85 et al. 2021). Consequently, there is renewed focus on whether crop productivity, 86 sustainability and resilience can be enhanced through improvements to WUE (Hatfield 87 and Dold, 2019; Leakey et al. 2019; Bailey-Serres et al. 2019; Sales et al. 2021). 88 Intrinsic WUE (iWUE) is defined as the ratio of the rate of net photosynthetic 89 CO2 assimilation (AN) relative to stomatal conductance ( gs). Improving iWUE can be 90 achieved by increasing AN without a matching increase in gs, or by decreasing gs 91 without a matching decrease in AN. However, many studies of natural and engineered 92 genetic variation within a broad diversity of plant species have shown AN and gs to be 93 correlated (Leakey et al. 2019; Deans et al. 2020). Consistent with this expectation, in 94 C3 species, theory and experimentation have shown that a decrease in stomatal 95 density drives lower gs and improves iWUE, but often at the cost of lower AN (Wang et 96 al. 2016; Hughes et al. 2017; Mohammed et al. 2019; Caine et al. 2019; Dunn et al. 97 2019). So, de -coupling of AN and gs in transgenic plants is rare and, when it is 98 achieved, iWUE is often reduced rather than increased (Flexas et al. 2013). 99 Meanwhile, crop genotypes selected by breeders for greater iWUE have often 100 been found to be innately less productive, as part of a generally conservative growth 101 syndrome (Condon et al. 2004). Although successes have resulted from screening 102 stable carbon isotopes as a proxy to select for high iWUE in C 3 species (Rebetzke et 103 al. 2006), scepticism remains about the potential for meaningful improvement of WUE 104 without a concomitant hit on yield (Condon et al. 2004; Blum 2005; Sinclair 2012). 105 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Recent innovations have emerged that open the door for improving WUE and 106 productivity in C 4 crops through lower gs via reduction in stomatal density . These 107 include novel high-throughput phenotyping tools to capture stomatal density and WUE 108 traits that are accelerating the speed and scale of experimentation (Ferguson et al. 109 2021, Pignon et al. 2021, Xie et al. 2021). Importantly, unlike C3 crops, C4 feedstocks 110 possess a carbon concentrating mechanism that increases [CO2] in the bundle sheath 111 cells where the primary photosynthetic enzyme Ribulose-1,5-Bisphosphate 112 Carboxylase Oxygenase (RuBisCO) is located; resulting in concentrations significantly 113 greater than atmospheric [CO2] (von Caemmerer and Furbank 2003). Consequently, 114 the relationship between AN and intercellular [CO2] (ci) features a much steeper initial 115 slope and a sharper inflection point than that observed in C3 species (Leakey et al. 116 2009). Atmospheric [CO 2] ha s risen from 370 ppm in 2000 to 417 ppm in 2023 117 (https://gml.noaa.gov/ccgg/trends/). As a result, the ci at which photosynthesis 118 operates in C4 crops is very close to saturation, which raises it above the A/ci curve 119 inflection point (Leakey et al . 2006; Ghannoum, 2008; Markelz et al . 2011). This 120 means more CO2 is entering the leaf than is required to maintain AN and, theoretically, 121 gs could be reduced to increase iWUE with little (<2%) to no increase in the stomatal 122

of AN (Leakey et al. 2019). The magnitude of reductions in stomatal density 123 needed to achieve an optimal gs is unknown, but too large a reduction in stomatal 124 density would lower ci to the point that it would fall below the inflexion point on the A/ci 125 curve, where stomatal limitation to AN becomes substantial (Leakey et al. 2019). 126 Mechanistic crop modelling suggests that a genetic strategy that translates to a 127 reduction in gs by 20% would significantly increase yields of a C 4 feedstock, like 128 sorghum, with the greatest gains occurring in marginal land locations where yields are 129 currently low (Leakey et al. 2019). Overexpression of STOMATAL DENSITY AND 130 DISTRIBUTION 1 (SDD1) in maize provided evidence in support of this approach, 131 driving reduced stomatal density and gs, without a decrease in AN (Liu et al. 2015). 132 But, pleiotropy was observed, with the low stomatal density plants also having greater 133 photosynthetic capacity tha n WT. The consequences of over-expressing SDD1 for 134 development, productivity, and WUE at the whole-plant scale were not reported. 135 Whole-plant transpiration rates regulate passive nitrogen uptake from the soil 136 (Niu et al. 2007; Matsunami et al. 2010; Kunrath et al. 2020). Therefore, a potential 137 drawback of a genetic strategy to improve WUE via a reduction in gs is that nitrogen 138 flux in the transpiration stream might be reduced, which in turn would negatively impact 139 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint photosynthetic capacity and productivity. A significant fraction of leaf nitrogen is 140 invested in photosynthetic proteins, with C4 species allocating less nitrogen to Rubisco 141 than C3 species, but more nitrogen to other soluble proteins and thylakoid components 142 (Ghannoum et al. 2011). However, with ectopic expression of EPF1 in the C3 species 143 wheat, barley and rice, photosynthetic capacity was reported not to decline (Hughes 144 et al. 2017; Caine et al. 2019; Dunn et al. 2019). 145 Testing the proposed approach to developing C 4 crops with greater WUE is 146 aided by the discovery of a network of genes that regulate leaf epidermal cell fate and, 147 thereby, stomatal density in C 3 species, because their C4 orthologs can be used as 148 initial candidate genes for testing ( McKown and Bergmann 2020 ). Most studies that 149 have manipulated stomatal density in C 3 species have achieved this by 150 overexpressing the native form of EPIDERMAL PATTERNING FACTOR 1 (EPF1), a 151 negative regulator of stomatal development (Harrison et al. 2020), or down-regulating 152 EPIDERMAL PATTERNING FACTOR -LIKE 9 (EPFL9 or stomagen), a positive 153 regulator of stomatal development (Karavolias et al. 2023). The EPF family of secreted 154 signaling peptides function within the epidermal cell layer to regulate stomatal 155 patterning (Hara et al. 2007; Hunt and Gray, 2009) . Various EPFs fusions swapping 156 the loop and scaffold regions of EPF2 and EPFL9 in Arabidopsis demonstrated that 157 the loop region confers the functional specificity of EPFs (Ohki et al. 2011). EPF 158 peptides trigger a mitogen activated protein (MAP) kinase signaling pathway that 159 regulates the stability of SPEECHLESS (SPCH). SPCH is a basic helix -loop-helix 160 (bHLH) transcription factor that contributes to the determination of cell division and 161 fate transitions (Lau et al. 2014). MAP kinases phosphorylate and destabilise SPCH 162 preventing the initiation of stomatal lineage cells, consequently overexpressing EPF 163 genes negatively regulates stomatal density via a decrease in SPCH protein levels 164 (Kumari et al. 2014). Recent evidence from C 3 monocotyledonous species, such as 165 barley, rice, and wheat, suggests that despite substantial differences in leaf expansion 166 and stomatal development, native grass EPFs act in a similar way to the 167 dicotyledonous model Arabidopsis’ EPFs; regulating entry to and progression through 168 the stomatal cell lineage (Hughes et al. 2017; Mohammed et al. 2019; Caine et al. 169 2019; Dunn et al. 2019). On the other hand, the role of other regulators of stomatal 170 and leaf development are not always strictly conserved across species and few 171 stomatal development genes have been tested in C4 species (Liu et al. 2009; Raissig 172 et al. 2016; Rassig et al. 2017; Schuler et al. 2018; Hughes and Langdale 2022). 173 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Sorghum has high WUE and is well adapted to xeric environments (Rooney, 174 2014). It is an important model C 4 species, with significant contributions to the 175 bioeconomy as a feedstock for food, feed and industrial applications (Paterson et al. 176 2009; Morris et al. 2013; Castro et al. 2015). Various models have shown that in high-177 yielding environments, a reduced water use trait can protect yield during soil moisture 178 deficits (Sinclair et al. 2005; Truong et al. 2017). 179 This study tested two genetic designs to reduce stomatal density in sorghum, 180 in order to address the knowledge gap about how engineering reduced stomatal 181 density would impact photosynthetic physiology and whole -plant function in a C 4 182 species. In the first design , the native sorghum EPF1 (SbEPF1) was constitutively 183 expressed under control of the sugarcane ubiquitin 4 (Ubi4) promoter (Wei et al. , 184 2003). In the second design, a fusion element was synthesized that combined 185 elements of the sorghum orthologs of At EPF2 and AtEPFL9 (SbEPFsyn) and placed 186 under control of the Ubi4 promoter. Comparing sorghum events with reduced stomatal 187 density verses wildtype controls addressed whether reduced stomatal density: i) can 188 drive reductions in gs without significantly increasing stomatal limitation to AN; ii) drives 189 pleiotropic effects on leaf physiology or plant development ; iii) produces leaf-level 190 reductions in gs that scale to reduced whole-plant water use; and iv) alters whole-plant 191 biomass production. 192 193

194 Assembly of binary vectors 195 The leucine rich repeat, receptor like kinase ERECTA corresponding interacting 196 partner EPIDERMAL PATTERNING FACTOR (EPF), EPF1, is a negative regulator of 197 stomatal development in Arabidopsis (Hara et al . 2007). An ectopic expression 198 cassette was designed to mis -express EPF1 in sorghum, and was designated 199 pPTN1337. The sorghum homolog of AtEPF1 (NM_127657), 200 Sobic006G233600.1/SbiTx43006G248600.1, ORF was synthesized (GeneScript, 201 USA), which incorporated the gene model’s 5’ and 3’ UTR elements. The synthesized 202 ORF with its corresponding UTRs, was subcloned between the sugarcane Ubi4 203 promoter (Wei et al. 2003) and the 3’ UTR of cauliflower mosaic virus 35S transcript 204 (T35S). The derived expression cassette was subsequently cloned into the binary 205 vector of pPZP212 (Hajdukiewicz et al . 1994) and the resultant vector designated 206 pPTN1337 (Supp. Fig. 1a). 207 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint 208 A second vector was designed for the expression of a fusion peptide that comprises a 209 domain from STOMAGEN (EPFL9; Hunt et al. 2010, Kondo et al. 2010) and EPF2 210 (Hara et al . 2009). This fusion consist s of AA residues 26 -219 from the sorghum 211 homolog of AtEPF2 (NM_103147), Sobic006G104400.1/SbiTx43006G109800.1 that 212 resides downstream of residues 24 -37 of the sorghum homolog of STOMAGEN 213 (AtNP_193033.1), Sobic003G299800.1/SbiTx43003G4311400.1. The fusion element 214 is imbedded within the 5’ and 3’ UTR of sorghum EPF2 gene model (Supp. Fig. 2). 215 This element was synthesized (GenScript, USA) and subsequently subcloned 216 between the sugarcane Ubi4 promoter and T35S terminator. The expression cassette 217 was then cloned into pPZP212 (Hajdukiewicz et al. , 1994) and the derived binary 218 vector designated pPTN1338 (Supp. Fig. 3a). 219 220 The derived binary vectors were introduced into A. tumefaciens strain NTL4/pTiKPSF2 221 (Luo et al. , 2001) and the resultant transconjugants used to transform the grain 222 sorghum genotype Tx430 as previously described (Howe et al., 2006, Guo et al., 2015) 223 224 Initial genotyping and phenotyping of transgenic events 225 Progeny derived from selfed lineages of the obtained transgenic events were 226 assessed for: (1) presence of the plant selectable marker allele via an NPTII ELISA 227 assay according to the manufacturer’s instructions (Agdia Inc., Elkhart, IN) and (2) 228 phenotyped for changes in stomatal density by optical tomography (Xie et al. 2021, 229 see below for details). Phenotyping was performed on 15 independent, positive events 230 of SbEPF1 and eight independent, positive transgenic events of SbEPFsyn. Of these, 231 two independent events carrying the SbEPFsyn allele, with significantly reduced 232 stomatal density were selected for further characterisation (Figure 1a). 233 234 The subset of the transgenic events, including the two SbEPF syn events, were 235 characterized by both Southern blot and RNA gel blot analyses as previously 236 described (Howe et al. 2006, Mall et al. 2011). Here, total genomic DNA was digested 237 with the EcoRI for Southern blot analysis (pPTN1338 events), and the membranes for 238 both northern and Southern hybridizations were probed with a 32P-labeled 719 bp 239 element that carried a region of the fusion ORF of pPTN1338 (Supp. Fig.3b,c). For the 240 RNA gel blot analysis conducted on a set of pPTN1337 events ( Supp. Fig.1b), the 241 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint membrane was hybridized with an approximate 570 bp region of the SbEPF1 ORF 242 spanning into the 3’ UTR. 243 244 Experimental design for detailed evaluation of SbEPFsyn 245 Plants were planted, grown, and phenotyped within the greenhouse facility at 246 the University of Illinois at Urbana-Champaign (latitude 40.11°, longitude -88.21°). The 247 greenhouse conditions were set to a 16h photoperiod (7AM -11PM) with 248 supplementary light provided by high pressure sodium and metal halide growth lamps. 249 The target day/night temperature was set to 28/21°C. 250 Wildtype (WT/Tx430) and T2 transgenic lineages were sown directly into trays 251 of 4-cm deep cells filled with Sunshine™ organic germinating mix (SunGro, Agawam, 252 MA). At the three -leaf stage, presence of the transgene was verified in the manner 253 described previously. Additionally, T -DNA copy number analyses of NPTII was 254 performed relative to a known single-copy sorghum gene amplicon by iDNA genetics 255 (Norwich, UK) , which confirmed the homozygosity of the transgenic allele in the 256 respective progeny lineages going forward . 257 Ten replicate plants from both WT and the two independent events of SbEPFsyn 258 (ZG602-5-12b and ZG602 -6-13a) were transplanted into 17.5L pots containing a 259 known mass of Sunshine Mix #4 professional growing mix (SunGro, Agawam, MA). 260 The mass of each pot and the soil within each pot was recorded to later allow 261 calculation of volumetric relative soil water content (% rSWC), as previously described 262 (Ferguson et al. 2018). Apart from 9 days mid-growing cycle when water was withheld 263 to perform a “dry-down” experiment (details below), plants were kept well-watered and 264 supplemented with liquid Nature’s Source 3 -1-1 NPK fertiliser (Ball DPF LLC, 265 Sherman, TX) bi-weekly. To avoid potential spatial bias, the positions of the pots were 266 randomised within blocks of the greenhouse space every three days across the full 267 duration of the study, as well as every day during the “dry-down” portion of the study. 268 Phenotypic sample and data collection occurred in three phases. First, at the 269 sixth leaf stage, the three most recently fully expanded leaves (i.e. leaves 4, 5, and 6) 270 were assessed for stomatal density. At that time, l ight-saturated rates of leaf 271 photosynthetic gas exchange, photosynthetic capacity, nitrogen (N) content, and 272 specific leaf area were also measured on the youngest, fully-expanded leaf. Second, 273 at the ninth leaf stage, a “dry -down” experiment was performed by withholding water 274 for nine days from the plants of each genotype. Two days prior to withholding water, 275 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint whole-plant leaf area was assessed. During the “dry -down” experiment, whole-plant 276 water use and photosynthetic leaf gas exchange were measured daily. Lastly, at plant 277 maturity, above-ground biomass production was determined. 278 279 Leaf gas exchange measurements 280 When leaf six was recently fully expanded, the response of net photosynthetic 281 CO2 assimilation (AN) to the concentration of leaf intercellular CO2 (ci) was measured 282 using a LI-COR 6800 infrared gas exchange system equipped with a standard 6cm2 283 cuvette (LI-COR Inc., Lincoln, NE). Data collection occurred between 0800-1500, just 284 prior to harvesting tissue from the same leaves for other leaf physiological traits. 285 Environmental conditions were set at : 27°C, 65% relative humidity (RH), 1800 μmol 286 m-2 s-1 photosynthetic photon flux density (PPFD), 400 µmol mol-1 CO2 concentration, 287 and 400 µmol s -1 flow rate. After full photosynthetic induction was established, AN, 288 stomatal conductance (gs), and ci were recorded as the leaf was exposed to a series 289 of stepwise changes in sample CO2 concentrations of: 400, 200, 50, 150, 300, 400, 290 500, 600, 700, 800, and 1200 µmol mol-1. A custom R function was used for modelling 291 An-ci response curves following (von Caemmerer, 2000) to estimate the maximum rate 292 of carboxylation by PEPC ( Vpmax) and the asymptote of the AN-ci curve ( Vmax), as 293 described previously (Markelz et al. 2011). 294 During each day of the water withdrawal experiment, AN and gs were measured 295 everyday between 0830 -1300 on the youngest fully expanded leaf . Measurements 296 were performed using LI -6400 gas exchange systems (LI-COR Inc., Lincoln, NE) 297 equipped with a 2cm x 3cm LED cuvette, and conditions in the gas exchange cuvette 298 were as described for the AN-ci response measurements. 299 300 Leaf stomatal density, SLA, N content 301 A Nanofocus µsurf explorer optical topometer (Nanofocus, Oberhausen, 302 Germany) was used to assess stomatal patterning, as described previously (Haus et 303 al. 2015; Ferguson et al. 2021). When leaf six was recently fully expanded, four fields 304 of view (800 x 800 µm each in size) arranged in a transect between the mid -rib and 305 margin at a position midway along the length of leaves four, five and six, were scanned 306 at 20x magnification on both the abaxial and adaxial surfaces. 3D reconstructions were 307 converted to 2D grey -scale images for analysis. Stomatal density was determined 308 using the cell counter feature in ImageJ (Abràmoff et al. 2006) 309 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint At the same time, leaf discs were sampled from the sixth leaf for estimation of 310 specific leaf area (SLA) and tissue nitrogen ( N) content, as described previously 311 (Markelz et al. 2011). 312 313 Whole-plant water use 314 To estimate water consumption during the “dry-down” experiment, plants were 315 soaked to ~100% rSWC and subsequently not watered for nine days. Each pot was 316 weighed daily during this period. These data were used to calculate rSWC whilst 317 accounting for pot weight at the start of the experiment, and plant mass measured on 318 three replicates harvested on the day that water withholding started. 319 320 Above-ground biomass production 321 Two-days prior to the water withdrawal period, whole plant leaf area was 322 determined as the sum of the width multiplied by the length of every leaf. This non -323 destructive estimation of leaf area is highly correlated to conventional measurements 324 of leaf area in maize (Pearce et al. 1975). 325 At full maturity, plants from both watering treatments were harvested just above 326 the soil level and dried at 60°C for two weeks before being weighed. 327 328 Statistical analyses 329 To test for overall phenotypic differences from WT in both the initial screening 330 of all transgenic events, and the subsequent detailed evaluation of events ZG602 -5-331 12b and ZG602 -6-13a for SbEPF2 syn under well -watered conditions, a one-way 332 analysis of variance (ANOVA) comparison of means test was performed. To then 333 determine which events were significantly different from the WT, a post-hoc Tukey test 334 was performed. 335 To test for differences in genotype effects on stomatal density between leaf 336 positions (i.e. leaf four, five or six), and to test for differences in genotype effects on 337 above-ground biomass between watering treatments (i.e. full -watered at all times 338 versus plants that experienced the “dry -down” treatment), t wo-way fully factorial 339 ANOVA tests were performed. 340 To determine genotypic differences in the response of rSWC, gs, and AN to 341 declining water availability, two-way fully factorial ANOVA tests were performed where 342 time was treated as a repeated measure . Post-hoc Tukey tests were subsequently 343 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint performed to ascertain on which days and between which genotypes significant 344 differences occurred. 345 All ANOVA tests were performed using the base lm() function in R. Where 346 multiple sub-samples were measured within a replicate plant i.e. stomatal density from 347 multiple field of view per leaf, an average was calculated for the replicate plant and 348 this was the input for all statistical tests. Least-squares means and standard errors for 349 all groups from each test were computed using the lsmeans() function from the 350 lsmeans R package (Lenth, 2016). The least-square means and standard errors are 351 reported in the associated bar and line plots. Post-hoc Tukey tests were performed 352 using the HSD.test() function from the agricolae R package (Mendiburu et al. 2015). 353 All figures were produced using the ggplot2 R package (Wickham, 2009) with post-354 processing in Affinity Designer (Serif, Nottingham, UK). 355 356

357 Peptide alignments of Arabidopsis and Sorghum epidermal patterning factors 358 SbEPF1, SbEPF2, and SbEPF9 359 Global alignments of the sorghum gene models that encode for the epidermal 360 patterning factors, EPF1, EPF2, and EPF9, with their corresponding Arabidopsis 361 homologs reveal varying degrees of homology at the protein level. The sorghum and 362 Arabidopsis EPF L9 peptides share approximately 47% identity with 66% similarity 363 (Supp. Fig 4), with the highest degree of identity about the C -terminal region, about 364 the core motif of the protein. An alignment of the sorghum and Arabidopsis EPF1 365 peptides display about 25% identity, with 31% similarity, with the highest degree of 366 relationship also at the C-terminal region of the peptide (Supp. Fig. 5). The respective 367 EPF2 proteins show 28% identity and 41% similarity, and like the other epidermal 368 patterning factors, the shared alignments occur at the C-terminal region of the peptide 369 (Supp. Fig. 6). 370 371 Phenotypic and molecular screens of SbEPF1 and SbEPFsyn 372 Eight of the fifteen independent events of SbEPF1 had significantly reduced 373 abaxial stomatal density compared to WT. However, in all but one case (NN547-3-2-374 1, -26% relative to WT) the reductions in stomatal density were very modest (-8 to -375 15%; Fig. 1). By contrast , five of the eight EPFsyn events had significantly lower 376 stomatal density than WT, and all five had much greater reductions in stomatal density 377 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint (-34 to -71%; Fig. 1). Two independent events (ZG602-5-12b and ZG600-6-13a), each 378 carrying a single homozygous copy of the transgene and with similar reductions in 379 stomatal density compared to WT ( -43 and -45%), were selected for further 380 investigation at the T2 stage. These lines are referred to as “12b” and “13a”. 381 382 Stomatal patterning and light-saturated leaf gas exchange of EPFsyn 383 In both lines of EPF syn, stomatal density was confirmed to be significantly lower than 384 WT on both the abaxial (p <0.001) and adaxial leaf surfaces (p < 0.001) of fully-385 expanded leaves at positions four, five and six on the main culm (Fig. 2). The average 386 reduction in SD was greater on the adaxial surface (-61% in line 12b, -36% in line 13a) 387 than on the abaxial surface (-43 % in line 12b, -30% in line 13a), and this effect was 388 consistent across leaf position s. All genotypes had significantly greater stomatal 389 density on the abaxial versus adaxial leaf surface. 390 Steady-state, light-saturated gs was significantly lower in EPF syn compared to 391 WT, with line 12b showing a stronger effect ( -32 %, p<0.01) than line 13a ( -18 %, 392 P<0.05; Fig. 3a). In line 12b, the stronger reduction in gs was accompanied by a 393 reduction in AN compared to WT (-22 %, p<0.01; Fig. 3b). For both lines of EPFsyn, the 394 reduction in gs led the ratio of AN to gs, (i.e., iWUE) to be significantly greater than for 395 WT (20% for line 12b, 13% for line 13a; p<0.01; Fig. 3c). 396 The observed changes in light -saturated gas exchange of EPF syn versus WT 397 were the result of changes in both stomatal and mesophyll limitations to 398 photosynthesis. As expected, stomatal limitation to AN was low (0.04; Fig. 4a) in WT, 399 and was unchanged in line 13a (0.04; Fig. 4 c). This corresponded to the operating 400 point of photosynthesis (blue dot) remaining above the inflection point on the A/ci curve 401 when gs was more moderately reduced in line 13a compared to WT. In line 12b, with 402 its stronger reduction in stomatal density, stomatal limitation to AN was greater (0.08; 403 Fig. 4b) than WT and the operating point of photosynthesis was at the inflection point 404 on the A/ci curve. EPFsyn plants of both lines had lower photosynthetic capacity than 405 WT as a result of significantly reduced apparent capacity for carboxylation by PEPC 406 (Vpmax; -32 in line 12b, -21 % in line 13a , p<0.01 ; Fig. 4d ) and also a marginally 407 significant reduction in the combined apparent capacity for carboxylation by Rubisco 408 and PEP regeneration by PPDK (Vmax) in line 12b (-20 %, p=0.11; Fig. 4e). There were 409 no significant differences between EPF syn lines and WT in SLA (p = 0.25; Fig. 4 f) or 410 leaf N concentration (p = 0.26; Fig. 4g). 411 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint 412 Interactions between leaf gas exchange, whole plant water use and drought stress413 Relative soil water content (rSWC) was significantly greater in pots of both 414 EPFsyn lines compared to WT as soon as day 2 of the dry-down experiment (Fig. 5a). 415 This water saving increased in magnitude until day 6, at which point WT plants had 416 almost exhausted the available soil water and their rate of soil drying slowed 417 dramatically. Since initial rates of soil drying were 30 -34% lower in EPF syn lines 418 compared to WT, they were sustained for longer before water supply became limiting, 419 only displaying a slight reduction in the rate of water use on day 9, at which point pot 420 soil moisture was almost exhausted for EPF syn and WT (Fig. 5a). Considering the 421 potential influence of plant size on rates of water use, a trend towards lower total leaf 422 area (7-12%) in the EPFsyn lines compared to WT was observed at the beginning of 423 the dry-down experiment, but the effect was not statistically significant (p = 0.60; Supp. 424 Fig. 7). 425 During the first five days of the dry-down experiment, when high rates of water 426 use were sustained in all pots, gs was 17 and 24% lower in the EPFsyn lines compared 427 to WT (p<0.05; Fig. 5b). This was accompanied by more modest average reductions 428 in AN in line 12b (-10 %, p=0.07) and line 13a ( -14 %, p<0.05) (Fig. 5c). On day 6, a 429 large and rapid decline in AN and gs occurred in WT, while leaf gas exchange declined 430 much more slowly in the EPF syn lines (Fig. 5b,c). As a result, gs and AN were 431 substantially greater for the EPFsyn lines compared to WT from day six through day 432 eight (Fig. 5b,c). The difference in drought stress experienced by the plants was 433 visually apparent with the WT being significantly wilted at the end of the “dry -down” 434 experiment while the EPFsyn lines retained full turgor (Fig. 6a). 435 The short period of drought stress relative to the overall growing period did not 436 significantly alter biomass production (p=0.65; Fig. 6b). Across both watering regimes, 437 both EPFsyn lines had slightly shorter internode lengths and overall heights (Fig. 6 a), 438 there was no significant difference among the genotypes in total dry, above-ground 439 biomass at maturity (p = 0.35; Figure 6b). A pleiotropic response of impaired panicle 440 and flower development were consistently observed in the EPFsyn lines, resulting in 441 significantly reduced seed production (Fig. 7). Occasionally, some leaves of EPFsyn 442 lines, but not WT, would exhibit a chlorotic strip that was 2-4cm in width and contained 443 almost no fully-developed stomata (Supp. Fig. 8). 444 445 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint

446 This study contributes to our understanding of how engineer ed reductions in 447 stomatal density impact leaf and whole -plant WUE in a model C 4 species, sorghum, 448 an important feedstock for the bioeconomy (Silva et al. 2022). Ectopic expression of 449 EPF1 has been shown to significantly reduce stomatal density and increase iWUE in 450 a number of C 3 species (Hughes et al. 2017, Dunn et al. 2019, Mohammed et al. 451 2019). In sorghum, however, ectopic expression of EPF1 resulted in slight reductions 452 in stomatal density (<-15%) in all but one transformation event (Fig. 1). By contrast, 453 ubiquitous expressi on of EPFsyn resulted in reductions of -34 to -71% in stomatal 454 density relative to WT (Fig. 1). Hence, deeper phenotypic characterizations were 455 performed on events carrying the SbEPF2 syn transgenic allele to address the 456 experimental objectives. Detailed evaluations of leaf gas exchange in two SbEPF2syn 457 events revealed improvements in iWUE compared to WT, while highlighting the 458 importance of achieving an intermediate reduction in stomatal density in order to lower 459 gs while still maintaining low stomatal limitation to AN (SL) (Figs. 3,4). Engineered gains 460 in iWUE drove reductions in whole-plant water use without causing a loss in above-461 ground biomass production (Figs. 5,6). In addition, the study revealed the potential for 462 pleiotropic effects on a number of developmental processes when engineering 463 changes in stomatal development by ubiquitous expression of this SbEPF2syn allele 464 (Fig. 4,7). These observations provide a framework for future research to genetically 465 enhance C4 plants in a manner that achieves the improvement in iWUE without an 466 increase in SL, while avoiding unwanted pleiotropic effects on development al 467 processes other than stomatal patterning. Achieving this goal is a high priority because 468 water-limitation is so important to global crop productivity, especially in regions where 469 agricultural production is vulnerable to climate variability, and because improving WUE 470 is such a valuable, if challenging, target for crop improvement ( Condon et al. 2004; 471 Blum 2005; Condon; Leakey et al. 2019). 472 473 Balancing reductions in gs with maintenance of stomatal limitation to AN 474 Analysis of A/ci curves can be used to quantify SL, which describes how much 475 lower the observed AN is compared to a theoretical AN where there is no resistance 476 for CO2 entry into the leaf i.e. ci equals the atmospheric [CO2] (Farquhar and Sharkey 477 1982; Long and Bernacchi 2003). One of the two SbEPFsyn events selected for detailed 478 characterization displayed changes in stomatal patterning and function that were close 479 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint to ideal. When comparing the light-saturated leaf gas exchange of line 13a to WT, 480 reduced stomatal density (-45%) was associated with lower gs, (-18%) and greater 481 iWUE (+13%; Figs. 2,3). This outcome resulted first from WT sorghum having low SL 482 (0.04; Fig. 4 a), as expected for a C 4 crop under contemporary [ CO2] of >410 ppm 483 (Leakey et al. 2019). Second, SL remained unchanged in line 13a (0.04) i.e. AN was 484 not reduced when lower stomatal density and gs limited diffusion of CO2 into the leaf 485 because the photosynthetic operating point remained above the inflection point of the 486 A/ci curve (Fig s. 3,4c). By contrast, SbEPF2syn event 12b had a slightly stronger 487 phenotype, where a greater reduction in stomatal density ( -69%) and gs (-32%) 488 resulted in greater stomatal limitation to A N (0.08) than in WT (0.04; Figs. 3,4b). This 489 resulted from the operating point of photosynthesis being at a lower ci, on the inflection 490 point of the A/ci curve, and contributed to AN of event 12b being lower (-22%) than WT 491 (Fig. 4b). These results validate the prediction, based on a leaf gas exchange model, 492 that reducing gs to an intermediate degree can enhance iWUE with very little to no 493 penalty to carbon (C) gain (Leakey et al. 2019). It is important to emphasize that the 494 steep inflection point of the C 4 A/ci curve means that improving iWUE without a 495 negative trade -off on photosynthetic C gain in sorghum, maize, sugarcane, 496 miscanthus and other C 4 grasses will depend on precisely tuning the reduction in 497 stomatal density to achieve greater iWUE without gs and AN. Under the growth 498 conditions used here, the optimal reduction in stomatal density lay somewhere 499 between the stomatal phenotypes observed in SbEPF2syn events 12b and 13a. As 500 future atmospheric [ CO2] continues to rise, progressively greater reductions in 501 stomatal density will be needed to maintain the photosynthetic operating point at the 502 optimal location just above the asymptote of the A/ci curve. 503 The results of this study suggest further effort should be applied to enhance 504 WUE in C4 crops by reducing stomatal conductance. While this study provides initial 505 proof-of-concept for achieving that goal by reducing stomatal density, approaches to 506 reduce stomatal complex size and/or the dynamic control of stomatal aperture by 507 guard cells are also exciting possibilities for which the foundation is being laid (Des 508 Marais et al. 2014, Nunes et al.2023). Being able to avoid an increase in SL while 509 increasing iWUE in a C 4 species is a distinct result from what is expected and often 510 observed in C3 species (Leakey et al. 2019). In C3 crops, the lack of a photosynthetic 511 carbon concentrating mechanism means that the A/c i curve is less steep, and 512 photosynthesis is not expected to be CO2-saturated today or later this century (Leakey 513 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint et al. 2006). Consequently, a modest loss in photosynthetic carbon gain under well-514 watered conditions results from engineering lower gs to achieve enhanced iWUE (e.g. 515 Yoo et al. 2010, Franks et al. 2015, Hughes et al. 2017, Caine et al. 2019). However, 516 crop modelling suggests this is a cost that is likely worthwhile for C 3 crops growing in 517 regions that are consistently water -limited (Leakey et al. 2019). Meanwhile, t he 518 possibility of avoiding th e negative trade-off between water savings and carbon gain 519 may allow low-gs/high-WUE C4 crops to enhance productivity across a wide range of 520 growing conditions from consistently water -limited to only occasionally water -limited 521 environments (Leakey et al. 2019). However, as described below, significant 522 challenges remain to be addressed to meet that goal. 523 524 Pleiotropic effects on mesophyll capacities for photosynthesis from ubiquitously 525 expressing SbEPFsyn 526 In addition to the effects of gs, SL can be influenced by change s in 527 photosynthetic capacity that alter the shape of the A/c i curve (Markelz et al. 2011). 528 Both SbEPFsyn events had lower apparent Vpmax than WT, which corresponds to a 529 shallower initial slope on the A/ci curve, and makes the inflexion point shift to greater 530 ci (Fig. 4). In event 12b, lower apparent Vpmax (-32 %) combined with a stronger 531 reduction in gs to cause the increase in SL (0.08) relative to WT (0.04). In addition, 532 there was a marginally significant (p=0.11) reduction in apparent Vmax in event 12b, 533 which corresponds to a lower asymptote on the A/c i curve. These reductions in the 534 mesophyll capacities for photosynthesis (apparent Vpmax and apparent Vmax) combined 535 with the greater SL to explain the lower light -saturated AN in event 12b compared to 536 WT (Fig. 4). 537 In event 13a, the pleiotropic effects of SbEPFsyn on mesophyll capacities for 538 photosynthesis were more moderate than in event 12b. Above the inflection point of 539 the A/ci curve, where CO2 supply is saturating, AN is limited by Vmax i.e. a combination 540 of Rubisco and phosphoenolpyruvate carboxylase capacity (von Caemmerer 2000). 541 The more moderate reduction in gs compared to WT, in event 13a, meant the operating 542 point of photosynthesis remained in this region just above the inflection point of the 543 A/ci curve (Fig. 4c) . Consequently, the lower apparent Vpmax (-21%) in event 13a 544 compared to WT had no consequences for A N. However, a reduction in Vpmax is still 545 generally undesirable from a crop performance perspective because, during drought 546 stress, when stomata will close and ci drops, photosynthesis will be operating on the 547 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint initial slope of the A/ci curve (i.e. below the inflection point) and thus a lower Vpmax will 548

in reduced carbon gain. Therefore, as atmospheric [ CO2] continues to rise, the 549 operating point of photosynthesis will continue to shift to greater ci, and photosynthesis 550 will only be operating on the initial slope of the A/c i curve during severe droughts 551 (Leakey 2009, Markelz et al. 2011). So, pleiotropic effects on Vpmax,, while not ideal , 552 will gradually become less of a concern in the future. 553 Apparent Vpmax is a measure of the steepness of the initial portion of the A/c i 554 curve, which is classically defined as the apparent carboxylation capacity of PEPC 555 (von Caemmerer 200 0). PEPC catalyzes the initial carboxylation reaction of C 4 556 photosynthesis in the mesophyll cells (Kanai and Edwards, 1999) . The activity of 557 PEPC is strongly correlated to leaf nitrogen content, as demonstrated recently in 558 sorghum (Khan et al . 2020), where nitrogen assimilation is critical for allowing 559 maximum photosynthesis. Moreover, in sorghum, maize, and rice it has been shown 560 that transpiration facilitates passive nitrogen flux, with consequences for leaf nitrogen 561 content (Niu et al. 2007; Matsunami et al. 2010; Kunrath et al. 2020). As such, it has 562 been hypothesised that reducing transpiration to improve WUE might reduce nitrogen 563 uptake and leaf nitrogen content (Hepworth et al. 2015), with potential consequences 564 for the biochemical capacity of photosynthesis. However, leaf nitrogen content per unit 565 leaf area was not significantly different between SbEPFsyn and WT (Figure 4g). 566 Therefore, the observed reduction in Vpmax is unlikely to be a function of this 567 phenomena. 568 Alternatively, the reduced photosynthetic capacity in SbEPFsyn plants might 569 have resulted from a signal transduction pathway triggered by expressing SbEPFsyn 570 ubiquitously i.e. signaling operating in parallel to the epidermal gene network driving 571 reduced stomatal density. In Arabidopsis, while EPF1 and EPF2 expression is focused 572 in epidermal cells, EPFL9 is expressed in mesophyll cells and interacts with targets 573 including light-induced factors regulating photomorphic growth (Hunt et al. 2010, 574 Kondo et al. 2010, Wang et al. 2021). Leaf capacities for photosynthesis, gas 575 conductance and hydraulic conductance need to be tightly coupled to optimize the 576 interplay between carbon and water relations ( Flexas et al. 2013), but the genetic 577 underpinnings governing this physiological coordination are not well understood. The 578 potential role of EPFs in this process would be consistent with changes in 579 photosynthetic capacity that have been observed i n some studies where expression 580 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint of native stomatal patterning genes have been modified (Liu et al. 2015, Franks et al. 581 2015, Caine et al. 2019, Dunn et al. 2019). 582 In addition to the influence of PEPC carboxylation capacity, t he initial slope of 583 a C4 A/ci curve (i.e. apparent Vpmax) can also vary in response to changes in mesophyll 584 conductance, which itself is determined by the resistance to CO2 diffusion through the 585 internal airspaces of the leaf and then in the liquid phase from the point of dissolving 586 in the apoplast to the point of the initial carboxylation in the mesophyll cell (Cousins et 587 al. 2020). A decrease in apparent Vpmax could, therefore, be driven by a decrease in 588 one or both of these components of mesophyll conductance. Such a response would 589 be consistent with the strong positive correlation between adaxial stomatal density and 590 mesophyll conductance observed across diverse C 4 grass species (Pathare et al . 591 2020). Plants with a greater number of adaxial stomata per unit area demonstrated 592 higher rates of mesophyll conductance due to an increase in the mesophyll surface 593 area exposed to intracellular air spaces, which creates additional routes for CO 2 594 diffusion to the initial site of fixation by PEPC. A pleiotropic effect of SbEPFsyn on 595 mesophyll airspace development rather than photosynthetic biochemistry would be 596 consistent with the general role of EPFs in cell fate determination and tissue 597 development. For example, in wheat events ectopically expressing EPF1, leaf 598 porosity was reduced alongside reductions in stomatal density and stomatal 599 conductance (Lundgren et al. 2019). Further experimentation will be needed to 600 determine if these anatomical changes are sufficient to alter mesophyll conductance, 601 and to test if they are occurring in low -stomatal density C 4 plants. Genes such as 602 SCARECROW (SCR) and SHORTROOT (SHR) play roles in regulating both 603 mesophyll and stomatal patterning in grasses (Schuler et al. 2017; Hughes et al. 604 2023). So, pathways that determine anatomical limitations to stomatal and mesophyll 605 conductance can be connected. Further work will be needed to determine if mis -606 expression of native or synthetic EPFs can regulate both of these aspects of leaf 607 development and function. It has been suggested that transgenes producing lower gs 608 could be stacked with additional transgenes that confer greater mesophyll 609 conductance, for example by making mesophyll cell walls less of a barrier to CO2 610 transport, thereby creating a positive synergistic effect on iWUE (Pathare et al. 2023). 611 612 Pleiotropic effects of ubiquitously expressing SbEPFsyn on plant development 613 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint An additional and more significant off -target effect of SbEPFsyn was observed 614 (Fig 7) on reproductive development, which may relate to interactions between EPF 615 and ERECTA proteins. Bioactive EPF2 peptides are known to bind and regulate 616 ERECTA to govern the division of protodermal cells into either pavement cells or 617 stomatal complexes (Zoulias et al. 2018). ERECTA-family receptor kinases coordinate 618 stem cell functions between the epidermal and internal layers of the shoot apical 619 meristem, and they are demonstrated to regulate floral patterning, fertility, and organ 620 identity in addition to stomatal patterning Arabidopsis (Shpak et al. 2003; Cai et al. 621 2017; Kimura et al . 2018). So, it is possible that the ubiquitous expression of 622 SbEPFsyn may have perturbed equivalent pathways in sorghum, leading to impaired 623 panicle and flower development (Fig . 7). If so, enhancing iWUE in sorghum while 624 avoiding pleiotropic effects on photosynthetic capacity and seed production might be 625 achieved by the use of tissue specific promoters that can limit expression of EPF syn 626 to the epidermis during early phases of leaf development. 627 628 Whole-plant biomass production, water use, and drought avoidance 629 The overall biomass production of SbEPFsyn plants was equivalent to that of 630 WT (Fig. 6). As described above, this appears to have been due to the pleiotropic 631 effects of expressing SbEPFsyn being focussed on developmental processes, and 632 under conditions of ample water supply, the anatomical consequences had mild (event 633 12b) to no effect (event13a) on photosynthetic carbon gain. 634 Total plant water use is a function of both the rate of water use per unit leaf 635 area and the total leaf area of the plant. The rate of water use by the two SbEPFsyn 636 events was -30 and -34% lower than WT (Fig. 5a). Most of the water savings can be 637 attributed to the reduction in gs, which averaged -20 and -23% lower in the two 638 SbEPFsyn events compared to WT, when averaged across all the dates of 639 measurement on which water supply was not limiting (Fig. 5b). However, it seems 640 likely that the -5 and -10% changes in total plant leaf area of the SbEPFsyn events 641 compared to WT also contributed to lower rates of water use, even if they were not 642 resolved as statistically significant (Supp. Fig. 7). 643 When water supply was withheld in the dry -down experiment, differences in 644 rates of water use meant that SbEPFsyn plants took nine days to exhaust the water 645 supply in their pots versus six days for WT (Fig. 5a). When the water supply ran out 646 for WT plants it triggered a substantial and rapid drop in gs and AN (Fig. 5b,c). It is 647 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint noteworthy that gs and AN declined gradually between days six to nine in the 648 SbEPFsyn events, compared to very abruptly decrease between days five and six in 649 the WT (Fig. 5b,c) . This is consistent with the interpretation of the A/c i curve data 650 described above i.e. under mild, initial drought stress the photosynthetic operating 651 point of SbEPFsyn plants required less of a drop in gs and c i to sit at or below the 652 inflection point of the A/c i curve, leading to a modest loss of C gain. Nevertheless, 653 these experimental results support the notion that a low stomatal density, low gs, high 654 iWUE strategy in a C4 crop results in greater carbon gain over the dry-down period as 655 a whole, which may enhance photosynthetic carbon gain and biomass production in 656 locations where water supply limits productivity (Leakey et al. 2019). It is important to 657 note that drought stress generally develops more rapidly and severely in pot 658 experiments than under field conditions. For example, d ry-down of the large soil 659 volume at field locations in the Central U.S. can take weeks rather than a few days to 660 develop (Leakey et al. 2004; Markelz et al. 2011; sorghum drought). In addition, the 661 dynamics of dry-down and re-wetting cause plant drought stress events to vary with 662 soil type and climatic conditions . So, field trials and crop modelling will be needed to 663 quantify the optimal phenotype of weak versus strong reductions in stomatal density 664 and gs across a range of growing conditions. 665 666 Conservation of EPF function across C3 and C4 lineages 667 In Arabidopsis, overexpressing the native EPF1 reduces stomatal density by 668 an average of 5 3% across >10 independent events (Hara et al . 2007). 669 Overexpressing, the barley EPF1 ortholog in Arabidopsis also produces a substantial 670 reduction (42% in barley across two events ) in stomatal density and overexpressing 671 the rice EPF1 ortholog in the epf2 Arabidopsis mutant restores WT stomatal density 672 (Hughes et al. 2017; Caine et al. 2019). This highlights the conserved functionality of 673 EPF1 across the dicot and C3 monocot functional types. Accordingly, overexpression 674 of the native EPF1 genes in barley, rice, and wheat achieves significant reductions in 675 stomatal densities that are like those or greater than those achieved via AtEPF1 676 overexpression in Arabidopsis , i.e., 52% average reduction in barley across two 677 events, 45% average reduction in rice across three events, and 70% average 678 reduction in wheat across two events (Hughes et al. 2017; Caine et al. 2019; Dunn et 679 al. 2019). The minor impact on stomatal density in response to the overexpression of 680 SbEPF1 in this present study (Fig. 1a), compared to what has been observed in barley, 681 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint rice, and wheat, is consistent with the possibility of functional divergence or 682 redundancy of EPF1 in sorghum and possibly other C4 grasses. Essential 683 developmental processes are often maintained through functional redundancy, where 684 two or more genes essentially perform the same function, such that manipulating the 685 expression of one of those genes has little or no effect on the prevailing phenotype 686 (Nowak et al. 1997). Gene and genome duplication events can enhance the likelihood 687 of gene functional redundancy as paralogous genes that overlap in functionality are 688 generated (Lee et al. 2014). Moreover, non -homologous genes can acquire similar 689 functions as species and clades diverge (Kafri et al . 2009). The hallmark Kranz 690 anatomy in C4 grasses is distinct from the leaf structure of C3 grasses, highlighting the 691 possibility for the evolution of functional redundancy and/or novel function acquisition 692 across these lineages. Indeed, the recent study of (Hughes et al. 2019) demonstrates 693 that Kranz cell patterning in maize is regulated in part by the redundant copies of the 694 SCARECROW 1 (SCR1) gene that play s different roles in the root and shoot. 695 Moreover, phylogenetic divergence in stomatal characteristics between C 3 and C 4 696 grasses has been observed to mirror the evolution of the C 4 photosynthetic pathway 697 and local adaption (Taylor et al. 2012; Lundgren et al. 2014). Our results regarding 698 SbEPF1 highlight the necessity of elucidating the genetic networks that underpin 699 stomatal development in C4 grasses to understand how they differ between C3 dicots 700 and grasses. The focus in the current experiments on understanding the downstream 701 effects on carbon and water relations of reduced stomatal density mean that 702 understanding the molecular mechanism by which EPF syn operated is beyond the 703 scope of the current study. This is just one of many knowledge gaps remaining about 704 the genetic basis for stomatal development in C4 grasses that need to be addressed. 705 706

707 In summary, this study provides support for prediction from modelling studies 708 that engineering to reduce stomatal conductance can improve iWUE and act to lower 709 plant water use without reducing biomass accumulation of a model C4 crop. However, 710 it also highlights: (1) the potential for pleiotropic effects on a range of developmental 711 when a mobile signaling peptide is expressed ubiquitously; and (2) the currently limited 712 understanding of the genetic basis for stomatal development in C 4 grasses. These 713 knowledge gaps will need to be addressed and more sophisticated engineering 714 strategies and/or additional genes targeted in order to retain the desirable stomatal 715 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint phenotypes observed here without the negative consequences of pleiotropic effects 716 on the agronomics of the crop. 717 718

719 We thank Mike Masters for assistance with elemental analysis. We thank the 720 UIUC Plant Biology and Crop Sciences greenhouse staff for support. The information, 721 data, or work presented herein was funded in part by the Advanced Research Projects 722 Agency-Energy (ARPA-E), U.S. Department of Energy, under Award Number DE-DE-723 AR0000661. No conflicts of interest are declared. 724 725

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Molecular control of stomatal 1002 development. Biochemical Journal 475, 441–454. 1003 1004 1005 1006 1007 1008 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 Figure Legends 1024 Figure 1. Stomatal density of independent transgenic events of (a) SbEPF1 and (b) 1025 SbEPFsyn. The events that displayed significantly different stomatal densities 1026 compared to the WT (according to a post-hoc Tukey test following a one-way ANOVA) 1027 are denoted via red asterisks. The independent events of EPFsyn that were carried 1028 forward for further investigation are highlighted in bold and underlined (ZG602-5-12b 1029 and ZG600-6-13a). 1030 1031 Figure 2. Representative micrographs of the abaxial leaf surface of : (a) WT, (b) 1032 ZG602-5-12b and (c) ZG600-6-13a, along with the associated (d) abaxial stomatal 1033 density and (e) adaxial stomatal density at three leaf positions on the main culm (three, 1034 four and five) for each genotype. Bars represent least square means of stomatal 1035 density for each grouping, where the errors bar represent the associated standard 1036 errors. For the abaxial and the adaxial surface, the p-values from each term in a two-1037 way ANOVA with an interaction term are inset. 1038 1039 Figure 3. Light-saturated (a) stomatal conductance ( gs), (b) net photosynthetic 1040 assimilation of CO2 (AN), and (c) Intrinsic water-use efficiency (iWUE) of the sixth leaf 1041 on the culm when it was the youngest fully expanded leaf of WT, ZG602-5-12b and 1042 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint ZG600-6-13a. Bars represent least square means and error bars represent associated 1043 standard errors. Significant differences between genotypes are denoted as * > 0.05, 1044 ** > 0.01, or *** > 0.001 1045 1046 Figure 4. Fitted average A-ci response curves of (a) WT, (b) ZG602-5-12b and (c) 1047 ZG600-6-13a, along with (d) apparent maximum rate of carboxylation by PEPC 1048 (Vpmax), (e) the asymptote of the AN-ci curve (Vmax), (f) specific leaf area (SLA), and (g) 1049 leaf nitrogen (N) content of the sixth leaf on the culm when it was the youngest fully 1050 expanded leaf. For A/c i curves, the mean fit is represented by the solid line and the 1051 standard error is denoted by the shaded area. The stomatal limitation to AN at ambient 1052 [CO2] (SL) is reported for each treatment. The operating point of photosynthesis at 1053 ambient [CO2] is shown as a blue dot on the A/c i curve. Bars represent least square 1054 means and error bars represent associated standard errors. The p -values from 1055 associated one-way ANOVA tests are inset. Where a significant effect was detected 1056 the differences between the transgenic lines and the WT according to post-hoc testing 1057 is shown. 1058 1059 Figure 5. The response of (a) percentage relative soil water content, (b) stomatal 1060 conductance (gs), and (c) net photosynthetic assimilation of CO 2 (AN) to a nine -day 1061 water withdrawal period. Points and errors bars represent least square means and 1062 standard errors, respectively. p-values associated with repeated measure ANOVAs 1063 are inset. Days where 12b and 13a were significantly different from WT for all traits 1064 are highlighted by red asterisks. The statistical results of pairwise tests between each 1065 transgenic line and the WT for a specific contrast of average gs and AN on the first five 1066 days of the experiment is shown by the inset bracket. 1067 1068 Figure 6. (a) Photographs of representative plants of WT, ZG602-5-12b and ZG600-1069 6-13a plants on day eight of the water withdrawal period. (b) Total dried above-ground 1070 biomass of all genotypes grown under water replete or subjected to the water 1071 withdrawal period. Bars represent least square means and error bars represent 1072 associated standard errors. p -values associated with each term from an associated 1073 two-way ANOVA are inset. 1074 1075 Figure 7. Photographs of representative panicles of WT and EPFsyn. 1076 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint 1077 Supplemental material 1078 Supplemental Figure 1. (a) The binary vector pPTN1337 carrying the ectopic 1079 expression cassette of the SbEPF1. Ubiquitin, UBI4, constitutive promoter from 1080 sugarcane. The 5’ & 3’ UTR elements of the gene model Sobic006G233600 delineate 1081 the SbEPF1open reading frame. The T35s polyAAA refers to the terminator of 1082 transcription from cauliflower mosaic virus. LB and RB refer to the left and right T -1083 DNA borders. While the aadA, ori/bom, and sta/rep indicate the positions of the 1084 backbone of the broad host range binary vector that include the bacterial selectable 1085 marker (aadA), for spectinomycin resistance, ori/bom origin of replication, and basis 1086 for mobility, and stability and second origin of replication motif (sta and rep). The plant 1087 selectable marker cassette resides proximal to the LB element. This cassette harbors 1088 the cauliflower mosaic virus 35s promoter, neomycin phosphotranferase II gene from 1089 E. coli (npt II) and is terminated by the T35s polyAAA. (b) Northern blot analysis on 1090 sorghum events (T1) carrying the SbEPF1 expression cassette (pPTN1337). Total 1091 RNA gel was hybridized with a 570 bp element that contained a downstream region of 1092 the SbEPF1 ORF, including some of the 3’ UTR. The expected 1.5 kb signal is 1093 indicated, the observed larger hybridization signal may be associated with the 1094 unprocessed transcript that still harbors the upstream intron associated with the UBI4 1095 promoter element. No signal was observed in the control Tx430 (WT: lane 11), likely 1096 to the relative low expression of SbEPF1, which is below detection with the 1097 hybridization assay. Lanes 1 -4: T1 individuals derived from event NN547 -3-2-1. 1098 Lanes 5-7: T1 individuals derived from event NN547-4-1-1. Lanes 8-10: T1 individuals 1099 derived from event TZ7-2-1. 1100 1101 Supplemental Figure 2. Diagrammatic representation of the translational product of 1102 the fusion peptide expression cassette of SbEPFL9/SbEPF2 present in the binary 1103 vector pPTN1138. The 14 amino acid residues shown in red at the N -terminal region 1104 of the fusion peptide from Sb stomagen (SbEPFL9) and the C -terminal amino acid 1105 residues 26 -219 from the SbEPF2 in black. The respective UTR’s from 1106 Sobic006G104400.1 are represented as Sb-5’UTR-EPF2 and Sb-3’UTR-EPF2. 1107 1108 Supplemental Figure 3. (a) The binary vector pPTN1338 fusion peptide expression 1109 cassette of SbEPLF9/SbEPF2, delineated by the 5’ & 3’ UTR from gene model 1110 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Sobic006G104400.1 and terminated by the T35s polyAAA. Other details of design 1111 match that of pPTN1337 and described in Supp. Fig. 1. (b) Southern blot and (c) 1112 northern blot analyses on sorghum events ZG600-6-13a, ZG6002-5-12b and ZG627-1113 3-30a of SbEPFsyn. (a): Southern blot restriction (EcoR1) digested total genomic DNA 1114 was hybridized with 719 bp element of the fusion ORF. The endogenous signal was 1115 observed in the control (WT) lane 4, at approximately 3.4 kb, while the events, lanes 1116 1-3, displayed varying two to four hybridizing loci demonstrating each event is an 1117 independent event. Lane + is approximately 10 pg of vector pPtN1138 digested with 1118 EcoR1. (b): northern blot analysis on total RNA from sorghum events ZG600 -6-13a, 1119 ZG6002-5-12b and ZG627-3-30a (lanes 1-3) and control (WT) lane 4. Membrane was 1120 hybridized with same probe used in the Southern. 1121 1122 Supplemental Figure 4. The global alignment of SbEPFL9, top strand, and AtEPFL9, 1123 bottom strand, reveal a 47% identity (black), and 66% similarity (blue). The loop motif 1124 is spanning residues 57-83, while the N-terminal fusion residues incorporated into the 1125 ORF of expression cassette that resides in the binary vector pPTN1338 is shown 1126 above residues 25-38. 1127 1128 Supplemental Figure 5. The global alignment of SbEPF1, top strand, and AtEPF1, 1129 bottom strand, reveal a 28% identity (black), and 41% similarity (blue). 1130 1131 Supplemental Figure 6. The global alignment of SbEPF2, top strand, and AtEPF2, 1132 bottom strand, reveal a 25% identity (black), and 31% similarity (blue). 1133 1134 Supplemental Figure 7. Total leaf surface area of wildtype, ZG602 -5-12b and 1135 ZG600-6-13a. Bars represent least square means and error bars represent associated 1136 standard errors. P-value from ANOVA testing the effect of genotype is inset. 1137 1138 Supplemental Figure 8. (a,b) Photographs of representative plant s featuring the 1139 occasionally observed phenotype of a short white band on a mature leaf, which (c) a 1140 representative optical tomography image shows to be a region almost entirely lacking 1141 stomata on the leaf epidermis. 1142 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Fig. 1 b a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Fig. 2 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint 3.4 kb 12.0 kb 1.5 kb b c Supp. Fig. 3 a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Fig. 4 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Fig. 5 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Fig. 6 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Figure 1 WT SbEPF1 SbEPFsyn preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint (a) (b) (c) Figure 2 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Figure 3 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Figure 4 0 10 20 30 40 50 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 ci (μmol mol-1)ci (μmol mol-1)ci (μmol mol-1) p = 0.11 (a) (d) (e) (f) (g) (b) (c) p = 0.25 p = 0.26 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 20 W T 12b 13a W T 12b 13a W T 12b 13a 12b 13a W T 40 60 80 100 Vpmax (μmol m -2 s -1 ) Vmax (μmol m -2 s -1 ) SLA (cm-2 g-1) 0 20 40 60 80 *** ** 0 75 150 225 300 375 450 0.0 0.5 1.0 1.5 2.0 N (% dry weight) 0 10 20 30 40 50 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 ci (μmol mol-1)ci (μmol mol-1)ci (μmol mol-1) p = 0.11 (a) (d) (e) (f) (g) (b) (c) p = 0.25 p = 0.26 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 20 W T 12b 13a W T 12b 13a W T 12b 13a 12b 13a W T 40 60 80 100 Vpmax (μmol m -2 s -1 ) Vmax (μmol m -2 s -1 ) SLA (cm-2 g-1) 0 20 40 60 80 *** ** 0 75 150 225 300 375 450 0.0 0.5 1.0 1.5 2.0 N (% dry weight) 0 10 20 30 40 50 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 ci (μmol mol-1)ci (μmol mol-1)ci (μmol mol-1) p = 0.11 (a) (d) (e) (f) (g) (b) (c) p = 0.25 p = 0.26 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 20 W T 12b 13a W T 12b 13a W T 12b 13a 12b 13a W T 40 60 80 100 Vpmax (μmol m -2 s -1 ) Vmax (μmol m -2 s -1 ) SLA (cm-2 g-1) 0 20 40 60 80 *** ** 0 75 150 225 300 375 450 0.0 0.5 1.0 1.5 2.0 N (% dry weight) 0 10 20 30 40 50 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 ci (μmol mol-1)ci (μmol mol-1)ci (μmol mol-1) p = 0.11 (a) (d) (e) (f) (g) (b) (c) p = 0.25 p = 0.26 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 10 20 30 40 50 AN (μmol m-2 s -1 ) 0 20 W T 12b 13a W T 12b 13a W T 12b 13a 12b 13a W T 40 60 80 100 Vpmax (μmol m -2 s -1 ) Vmax (μmol m -2 s -1 ) SLA (cm-2 g-1) 0 20 40 60 80 *** ** 0 75 150 225 300 375 450 0.0 0.5 1.0 1.5 2.0 N (% dry weight) SL = 0.04 SL = 0.08 SL = 0.04 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Figure 5 WT vs 12b p<0.05 WT vs 13a p<0.05 WT vs 12b p=0.07 WT vs 13a p<0.05 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Figure 7 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint (a) (b) WT 12b 13a Figure 6 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Figure 7 WT EPFsyn preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint Supp. Figure 8 (a) (b) (c) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 3, 2024. ; https://doi.org/10.1101/2024.02.01.578512doi: bioRxiv preprint