Local activity controls seed and yield The peptide hormone receptor CEPR1 functions in the reproductive tissue to control seed size and yield

1 The interaction of C -Terminally Encoded Peptides (CEPs) with CEP RECEPTOR 1 (CEPR1) 2 controls root growth and development, as well as nitrate uptake, but has no known role in 3 determining yield. We used physiological, microscopic, molecular and grafting approaches to 4 demonstrate a reproductive tissue-specific role for CEPR1 in controlling yield and seed size. 5 Independent Arabidopsis ( Arabidopsis thaliana ) cepr1 null mutants showed 6 disproportionately large reductions in yield and seed size relative to their decreased 7 vegetative growth. These yield defects correlated with compromised reproductive 8 development predominantly in female tissues, as well as chlorosis, and the accumulation of 9 anthocyanins in cepr1 reproductive tissues. The thinning of competing reproductive organs to 10 improve source-to-sink ratios in cepr1 , along with reciprocal bolt-grafting experiments, 11 demonstrated that CEPR1 acts locally in the reproductive bolt to control yield and seed size. 12 CEPR1 is expressed throughout the vasculature of reproductive organs, including in the 13 chalazal seed coat, but not in other seed tissues. This expression pattern implies that CEPR1 14 controls yield and seed size from the maternal tissue. The complementation of cepr1 mutants 15 with transgenic CEPR1 rescued the yield and other phenotypes. Transcriptional analyses of 16 cepr1 bolts showed alterations in the expression levels of several genes of the CEP-CEPR1 17 and nitrogen homeostasis pathways. This transcriptional profile was consistent with cepr1 18 bolts being nitrogen-deficient, and with a reproductive tissue-specific function for CEP- 19 CEPR1 signalling. The results reveal a local role for CEPR1 in the maternal reproductive 20 tissue in determining seed size and yield, likely via the control of nitrogen delivery to the 21 reproductive sinks.

Whether CEP-CEPR1 signalling plays a role in the growth of shoots has not been thoroughly 61 explored. Aboveground, the cepr1-1 mutant has been described as dwarfed, producing 62 smaller rosettes with pale green leaves and a shorter floral stem that over accumulates 63 anthocyanins (Bryan et al., 2012;Tabata et al., 2014). Since these traits are typical responses 64 to nitrogen deficiency (Vidal and Gutiérrez, 2008;Takatani et al., 2014), it could be reasonable to dismiss any cepr1 aboveground defects as simply the result of reduced nitrate 66 acquisition by the cepr1 roots. Our anecdotal observations of two cepr1 null mutants, 67 however, indicated that their yield was reduced much more dramatically than expected based 68 on their modest reduction in vegetative growth. In addition, we observed that the cepr1 69 mutants produced smaller seeds. These phenotypes appear inconsistent with an effect of 70 CEPR1 on nitrate uptake alone, given that wild-type plants grown at low nitrogen, or mutants 71 with impaired nitrate uptake, produce normally sized but fewer seeds such that their yield 72 losses are proportional to the decreases in vegetative growth (Schulze et al., 1994;Masclaux-73 Daubresse and Chardon, 2011).

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In contrast to plants with impaired root nitrate uptake, an impairment in the remobilisation of 75 assimilated nitrogen from vegetative to reproductive tissues leads to a reduction in both seed 76 size and yield (Guan et al., 2015;Li et al., 2015;Di Berardino et al., 2018;Moison et al., 77 2018). Smaller seeds also result from knocking out particular UMAMIT genes, which encode 78 transporters required for delivering assimilated nitrogen to seeds (Müller et al., 2015). It is 79 not known if CEPR1 signalling affects nitrogen mobilisation and delivery to reproductive 80 sinks, however the phenotypic similarities of cepr1 with mutants impaired in these processes 81 hint at this possibility, and suggest that CEPR1's control over seed size and yield extends 82 beyond its influence over root nitrate uptake. Supporting this hypothesis, we noted that 83 several Arabidopsis CEPs are expressed in reproductive tissues (Roberts et al., 2013), and 84 that rice (Oryza sativa) CEPs OsCEP5 and OsCEP6 are specifically expressed at defined 85 stages of reproductive development (Ogilvie et al., 2014;Sui et al., 2016). CEPR1 is 86 expressed throughout the shoot and root vasculature (Bryan et al., 2012;Huault et al., 2014;87 Tabata et al., 2014), suggesting roles in multiple tissues. 88 To explore a potential role for CEP-CEPR1 signalling in reproduction, we addressed several 89 questions. Firstly, what is the physiological basis for yield reduction in cepr1 mutants? 90 Second, can yield losses in cepr1 be restored by complementation with transgenic CEPR1 or 91 by manipulating nutrient allocation from the vegetative tissues? Third, is CEPR1 expressed in 92 reproductive tissues, and is its effect on yield controlled systemically via vegetative tissues or 93 locally in reproductive tissues? Finally, does CEPR1 regulate genes involved in nitrogen 94 homeostasis/nutrient mobilisation in the reproductive tissues?

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In this study, we demonstrate that CEPR1 has a specific role in reproductive tissues in the 96 promotion of fecundity, seed yield and size. The two cepr1 knockout mutants showed yield reductions of between 88% and 98%, which were associated with the production of smaller 98 seeds and a diminished number of reproductive units. These yield defects correlated with 99 poorly developed reproductive tissues as well as chlorosis and the accumulation of 100 anthocyanins in cepr1 reproductive tissues, all of which could be restored by transgenic 101 complementation with CEPR1. Bolt grafting and manipulation of nutrient allocation to 102 reproductive sinks showed that local CEP-CEPR1 signalling underpins the poor fecundity of 103 the cepr1 mutants. We found that CEPR1 expression in the reproductive organs occurred 104 specifically in the vasculature. Notably, CEPR1 expression in the seed was restricted to the 105 chalazal seed coat, the site where nutrients for seed filling are unloaded from the terminating 106 maternal vasculature (Müller et al., 2015). This result supported a local role for CEPR1 in the 107 control of seed size and yield through activity in the mother tissue. Finally, we used 108 transcriptional profiling of key marker genes to demonstrate a perturbation of nitrogen status 109 in cepr1 bolts. Collectively, the results reveal a role for CEP-CEPR1 signalling that involves 110 local activity in the bolt to control nitrogen mobilisation and delivery to reproductive sinks.

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CEPR1 controls vegetative growth, reproductive development and seed yield 113 We explored whether CEPR1 plays a role in vegetative and reproductive development by 114 examining two independent knockout mutant alleles in the No-0 and Col-0 backgrounds 115 (cepr1-1 and cepr1-3, respectively) (Tabata et al., 2014;Chapman et al., 2019). Both cepr1 116 knockout mutants displayed a ~30% retardation in rosette growth (Fig. 1, A-C). Since 117 vegetative leaves mobilise resources to the bolt and other reproductive tissues, we examined 118 whether loss of CEPR1 function affected yield. The cepr1 mutants displayed a reduction in 119 the growth of reproductive tissues (Fig. 1,D and E), and the number of reproductive units 120 (siliques, flowers and buds) on the main inflorescence was reduced by ~10% in cepr1-1 and 121 ~40% in cepr1-3 (Supplemental Fig. S1). The diminished reproductive capacity correlated 122 with a substantial reduction in the total seed yield per plant of ~88% and ~98% in cepr1-1 123 and cepr1-3, respectively (Fig. 1, F and G). The reduced seed yield resulted from a lower 124 seed number and a 12-25% reduction in seed weight (Fig. 1,H and I). In general, the limited 125 seeds produced by the cepr1 mutants were smaller and more diverse in size compared to 126 wild-type (WT) seeds (Fig. 1, J and K). These results demonstrate that CEPR1 knockout 127 reduces vegetative growth as well as seed size and yield.
The severe reductions in cepr1 seed yield (~88-98%) seemed disproportionate to the decrease   Since the loss of CEPR1 function affected flower development, we investigated whether 164 there were effects on yield per silique in cepr1 (Fig. 3). First, we assessed siliques of self-165 pollinated plants and observed that cepr1 mutants had a higher incidence of unfertilised 166 ovules, and of seeds that had aborted at various stages of development (Fig. 3, A and B). The 167 reduction in seed set per silique was particularly severe in cepr1-3, and worsened acropetally.  (Fig. 3D). In contrast to WT Col-0, cepr1-3 had maximal seed set in 175 the first silique, which decreased with increasing silique position (Fig. 3D). We observed nil 176 seed set from silique number 12 onwards in cepr1-3. For siliques with non-zero fecundity, the 177 average seed set was significantly lower in cepr1-1 and cepr1-3 compared to their respective 178 WT lines (Fig. 3, E and F). Moreover, the frequency of clearly fertilised and then aborted 179 seeds (i.e. late aborting seed) was higher in both cepr1 mutants compared to their respective 180 WT lines, and this phenotype was particularly severe in cepr1-3 (Fig. 3, G and H). 181 We conducted reciprocal crosses to determine whether the more severe seed set reduction in 182 cepr1-3 was due to a male and/or female reproductive defect (Fig. 3I). Pollen from cepr1-3 183 fertilized WT pistils without impairment, which suggested that there was no appreciable 184 decrease in cepr1-3 pollen viability. The reduced fecundity of the cepr1-3 pistil compared to 185 the WT held true regardless of pollen genotype. As mechanical pollination would overcome 186 pollen deposition defects resulting from asynchronous anther-gynoecium development in  To further explore this female fertility defect, we examined fertilisation frequency depending 190 on ovule position in the pistil. Such analysis helps distinguish between problems with pollen transmission and ovule-specific defects (Kay et al., 2013;Groszmann et al., 2020). As cepr1 192 mutants in both backgrounds had fewer ovules per pistil than WT (Supplemental Fig. S2), we 193 assessed seed set at normalised ovule positions within the carpels, from position zero (closest 194 to stigma) to position one (closest to gynophore) (Fig. 3, J and K). Compared to 195 cepr1-1 showed a similar, albeit more accentuated, position-dependent fertilisation pattern in 196 the pistil (Fig. 3J). As expected, ovules of WT Col-0 pistils showed a high and mostly 197 position-neutral fertilisation frequency (Fig. 3K). In stark contrast, the fertilisation rate in  One component of the yield decrease in cepr1 is a reduction in mature seeds per silique due 208 to a reduced ovule number, lower fertility rate and an increased incidence of seed abortion.

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In addition, cepr1 seeds that successfully progress to maturity are smaller than WT seeds, 210 further contributing to the lower yield. Normally plants with a reduced seed number tend to 211 compensate by having larger seeds, which is due to a greater proportional allocation of the 212 available nutrients being remobilised from the rosette (Bennett et al., 2012). Therefore, the 213 small mature seeds of cepr1 may reflect a deficiency in nutrient supply to the seeds. This 214 may arise due to its smaller source rosette and hence, a proportionately lower per seed 215 availability of nutrients; or alternatively, due to a reduced capacity of cepr1 plants to deliver 216 nutrients from the source rosette to the seed. To test these possibilities, we manipulated 217 nutrient allocation from the rosette (source) by thinning the number of competing 218 reproductive organs (sinks), to improve the source-to-sink ratio in favour of larger seeds in 219 the siliques that remained (Bennett et al., 2012). As expected, we found that seed size 220 increased in response to the thinning of WT plants, however, an increase in seed size did not 221 occur in cepr1-1 despite improving the source-to-sink ratio (Fig. 4). This result suggests that 222 CEPR1 is required for the redistribution and/or delivery of resources for seed filling, and in 223 doing so, controls seed size. 224 CEPR1 control of seed size depends on CEPR1 activity in the reproductive bolt 225 We undertook reciprocal bolt grafting between WT and cepr1-1 to elucidate if CEPR1 226 activity in the vegetative tissues or reproductive tissues determined seed size (  At maturity, we harvested the seeds and other dry bolt materials (i.e. stem, cauline leaves, 232 floral and silique material) to assess the effect of graft combination on seed size and yield. 233 We found that the smaller size of seeds produced by cepr1 bolts could not be rescued by 234 grafting to a WT stock (Fig. 5D). In contrast, there was no penalty to the seed size of WT 235 bolts when grafted to cepr1-1 stock (Fig. 5D). Furthermore, the distribution of seed size for 236 WT bolts was more uniform than for cepr1-1 bolts regardless of the stock (Fig. 5E). This 237 suggests that local CEPR1 activity in the bolt controls seed size and its uniformity. A weak, 238 compensatory effect of vegetative CEPR1 activity was observed for cepr1-1 bolts grafted 239 onto a WT stock (i.e. cepr1-1/WT) with mild improvements in seed size uniformity 240 compared to cepr1-1 homografts (Fig. 5E).

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Since the decrease in total seed yield of the cepr1-1 bolt could not be rescued by grafting to a 243 WT stock (Fig. 6A), a lack of CEPR1 activity in the bolt most likely limits yield in cepr1-1 244 plants. Moreover, there was no yield penalty to a WT bolt when grafted to a cepr1 stock, but 245 rather an apparent increase in total seed yield relative to the WT homografts (Fig. 6A). An harvest index of cepr1-1 slightly improved when grafted to a WT stock (increasing from ~37% to ~50% of WT homografts). Therefore, whilst a WT stock could partially compensate 254 for a lack of CEPR1 in the bolt, the bolt harvest index was primarily determined by CEPR1 255 activity in the bolt. These results, together with the seed size data, show that CEPR1 bolt 256 activity controls reproductive development, seed size, and subsequently total yield.  Therefore, nutritional limitation may account for the cepr1 defects in floral morphology, 300 lower fecundity, the high incidence of seed abortion and its smaller, more variable seed sizes.

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To investigate this, we harvested bolt and inflorescence tissues and surveyed the expression were also differentially expressed in the cepr1 mutants. CEP5 and CEP9 were strongly 308 upregulated (~32-fold), CEP2 was downregulated in cepr1-3 (~3-fold) and undetectable in 309 the No-0 background, and CEP1 expression was not significantly altered (Fig. 9A). The    Improving nutrient allocation by thinning the number of reproductive units resulted in the 369 expected increased seed size in WT plants, but not in cepr1 mutants. The differential effect 370 of thinning between WT and cepr1 is not due to the smaller cepr1 rosettes, because grafting 371 showed that the cepr1 rosettes support the flourishing of WT bolts. Moreover, this grafting 372 result implies that the cepr1 source rosettes are still able to sufficiently load nutrients such as 373 amino acids into the phloem for export to the developing bolts (Santiago and Tegeder, 2016).

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Therefore, the seeds of cepr1 plants that were thinned appear unable to receive the expected 375 allocation of surplus nutrient that clearly benefits the seeds of thinned WT plants.   Supplemental Table S1. were generated by crossing with WT Col-0 transformed with the complementation construct.

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Flowers from independent Col-0 lines harbouring the CEPR1 complementation construct 480 were emasculated and then pollinated using cepr1-3 pollen. F2 plants homozygous for the 481 cepr1-3 allele (i.e. no endogenous WT CEPR1) and carrying the CEPR1 transgene were 482 identified using PCR genotyping (see Supplemental Table S1 for primers).    Table S1 for genotyping primers used.    Table S1. List of oligos used.

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Quantification of (A) total seed yield, (B) bolt dry mass (minus seed), and (C) bolt harvest 632 index (the ratio of seed yield to total bolt biomass). Significant differences determined by 633 ANOVA followed by Tukey HSD test (alpha=0.05). Error bars show SE. n = 6-9 plants.