Genome-wide analysis of OVATE family proteins in cucumber (Cucumis sativus L.)

29 Background: OVATE family proteins (OFPs) are plant-specific proteins with the 30 conserved OVATE domain that regulating plant growth and development. Although 31 these OFPs have been studied in several species, the biological functions of this OFP 32 gene family remain largely unknown in cucumber ( Cucumis sativus L.). 33 Results: In this study, we identified 19 CsOFPs in cucumber. This CsOFPs are 34 distributed on seven chromosomes and can be divided into four subgroups. Most 35 CsOFP genes are expressed in reproductive organs although have different expression 36 patterns. Cis -elements analysis showed that there are six kinds of hormone response 37 elements in CsOFPs and exogenous gibberellin treatment leads to a ‘first increase then 38 decrease’ expression pattern of CsOFP7 , CsOFP11 and CsOFP12 . Ectopic expression 39 of CsOFP11 in Arabidopsis resulted in shorter and blunt siliques. 40 Conclusions: Together, these results indicated that CsOFPs may play important roles 41 in cucumber fruit development. 42


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OVATE gene was originally identified in tomato， in which a natural mutation in OVATE 47 resulted in fruit from round to pear-shaped [1]. One century ago, pear-shaped fruit form 48 in tomato was proposed to be controlled by a single recessive quantitative trait locus 49 (QTL), which was named pyriform (pr) [2][3]. Later, additional studies showed that pr 50 was co-segregate with the locus determining oblate-to oval-shaped fruit, pr was virus-induced gene silencing in cv. "Mytilini Round" caused its fruit to a more oblong 66 shape [9]. Some studies suggested that GS9 could interact with OsOFP14 and OsOFP8 67 to regulate rice grain shape and knockout of GS9 resulted in slender grains [10]. 68 Moreover, the interaction pair MuMADS1 and MaOFP1 are antagonistically regulated 69 by ethylene and might participated in the process of fruit ripening in banana [   AtOFP20, which have one intron (Fig. 1b). However, these intron-containing CsOFP 129 and AtOFP genes are distributed in different subgroups, except that the most closely 130 related AtOFP17 and AtOFP20 genes fall into the same subgroup. results showed that the OVATE domain contains two conserved motifs, in which they 135 shared some conserved amino residues ( Fig. 2a-b). These conserved amino residues 136 may be critical for protein-protein interactions between OFPs and other proteins. 138 To further study the temporal and spatial transcription patterns of the CsOFP genes in  In total, 11 hormone response cis-elements were identified in CsOFP promoter regions.

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The number of hormone response cis-elements was variable in each CsOFP promoter 157 region (Fig. 4a). There are ten cis-elements in CsOFP1b promoter region, but only one 158 for CsOFP8a and CsOFP16 (Fig. 4a). These cis-elements are related to responses of although had no significant morphological phenotypes compared to WT (Fig. 6a-b).

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The transgenic line #34 exhibited shorter silique lengths than other transgenic lines, 181 consistent with their highest expression of CsOFP11 ( Fig. 6c and g). And then the

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Recent studies revealed that several OFPs negatively control the fruit length. For 215 example, both OVATE and SlOFP20 regulate fruit length in tomato [1,21]. In melon 216 (Cucumis melo), six candidate genes in a QTL associated with shape on chromosome 217 8 include CmOFP13. In cucumber, the fruit shape QTL fs3.2 interval also contains an 218 OFP member CsOFP15a [21]. In this study, the transgenic plants also exhibited shorter 219 and wider siliques compared to WT plants when overexpression of CsOFP11 in 220 Arabidopsis (Fig. 6b-c). Although genetic evidence is limited, it seems likely that these 221 OFP genes regulate fruit length in several plant species. In addition, the transgenic 222 Arabidopsis overexpressing CsOFP11 also showed blunt siliques (Fig. 6f)

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In this study, we identified 19 CsOFPs in cucumber. 14 of these CsOFP members were 230 expressed in reproductive organs. When CsOFP11 transformed into the Arabidopsis, 231 the transgenic lines exhibited shorter but blunt siliques, indicating that these CsOFP 232 members may be involved to fruit development. In the future, it will be worth to 233 investigate the biological function of the CsOFPs in cucumber.  The female flower buds of 40-day-old cucumber were sprayed with 50uM GA3, and 266 they were harvested at 0h, 0.5h, 2h, 4h, 12h, and 24h, respectively. For each time point, 267 2 female flower buds were harvested as one biological repeat for RNA isolation.        CsOFP expression triggered by GA in female ower buds. (a-d) The cucumber female ower buds of 40day-old cucumber were sprayed with with 50uM GA3 and the expression level of CsOFP15b (a), CsOFP7 (b), CsOFP11 (c), and CsOFP12 (d) were examined at 0h, 0.5h, 2h, 4h, 12h, and 24h after treatment. The cucumber UBIQUITIN gene was used as an internal standard. Values are means ± sd of three biological