Bolting, which is a serious problem during lettuce (Lactuca sativa L.) production, is responsible for substantial annual yield and quality losses. Gibberellin plays a critical role in the regulation of lettuce bolting. Additionally, DELLA proteins negatively regulate the gibberellin signaling pathway. However, it is unclear if DELLA proteins are involved in the regulation of lettuce bolting. Therefore, in this study, we identified four DELLA-encoding genes in lettuce, including LsRGL1, which was highly expressed in the stem and negatively correlated with bolting. Knocking down this gene in lettuce promoted bolting, whereas its overexpression inhibited bolting and the biosynthesis of gibberellin and auxin. A transcriptome analysis revealed that genes involved in gibberellin and auxin biosynthesis and flowering were affected in the LsRGL1-overexpressing lines. The yeast two-hybrid and yeast one-hybrid assay results indicated that LsRGL1 can interact with LsGA3ox and the LsYUC4 promoter region. Considered together, the results of this study suggest LsRGL1 negatively regulates lettuce bolting. Furthermore, its function may depend on modifications to gibberellin and auxin levels mediated at the transcript and protein levels.

Leaf size and flatness directly affect photosynthesis and are closely related to agricultural yield. The final leaf size and shape are coordinately determined by cell proliferation, differentiation, and expansion during leaf development. Lettuce ( Lactuca sativa L.) is one of the most important leafy vegetables worldwide, and lettuce leaves vary in shape and size. However, the molecular mechanisms of leaf development in lettuce are largely unknown. In this study, we showed that the lettuce APETALA2 ( LsAP2 ) gene regulates leaf morphology. LsAP2 encodes a transcriptional repressor that contains the conserved EAR motif, which mediates interactions with the TOPLESS/TOPLESS-RELATED (TPL/TPR) corepressors. Overexpression of LsAP2 led to small and crinkly leaves, and many bulges were seen on the surface of the leaf blade. LsAP2 physically interacted with the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors and inhibited their transcriptional activation activity. RNA sequencing analysis showed that LsAP2 affected the expression of auxin- and polarity-related genes. In addition, LsAP2 directly repressed the abaxial identity gene KANADI2 ( LsKAN2 ). Together, these results indicate that LsAP2 regulates leaf morphology by inhibiting CIN-like TCP transcription factors and repressing LsKAN2 , and our work provides insights into the regulatory mechanisms of leaf development in lettuce.

Seeds are major vehicles of propagation and dispersal in plants. A number of transcription factors, including APETALA2 (AP2), play crucial roles during the seed development process in various plant species. However, genes essential for seed development and the regulatory networks that operate during seed development remain unclear in lettuce. Here, we identified a lettuce AP2 (LsAP2) gene that was highly expressed during the early stages of seed development. LsAP2 knockout plants obtained by the CRISPR/Cas9 system were used to explore the biological function of LsAP2. Compared with the wild type, the seeds of Lsap2 mutant plants were longer and narrower, and developed an extended tip at the seed top. After further investigating the structural characteristics of the seeds of Lsap2 mutant plants, we proposed a new function of LsAP2 in seed dispersal. Moreover, we identified several interactors of LsAP2. Our results showed that LsAP2 directly interacted with the lettuce homolog of BREVIPEDICELLUS (LsBP) and promoted the expression of LsBP. Transcriptome analysis revealed that LsAP2 might also be involved in brassinosteroid biosynthesis and signaling pathways. Taken together, our data indicate that LsAP2 has a significant function in regulating seed shape in lettuce.

2 Running title: LsAP2 regulates lettuce seed shape 3 4 Chen Luo*, Shenglin Wang*, Kang Ning, Zijing Chen, Yixin Wang, Jingjing Yang, 5 Meixia Qi, Qian Wang 6 7 Beijing Key Laboratory of Growth and Developmental Regulation for Protected 8 Vegetable Crops, Department of Vegetable Science, College of Horticulture, China 9 Agricultural University, Beijing 100193, China 10 11 *These authors contributed equally to this work. 12 Author for correspondence: wangq@cau.edu.cn 13 14 Date of submission: 2020/11/23 15 Number of tables: 0 16 Number of figures: 7 17 Number of supplementary tables: 6 18 Number of supplementary figures: 6 19 Number of supplementary data: 1 20 Word count: 4530 (Introduction: 840; Results: 2481; Discussion: 1145; 21 Acknowledgments: 64) 22 23

Domesticated crops with high yield and quality are frequently susceptible to pathogen attack, whereas enhancement of disease resistance generally compromises crop yield. The underlying mechanisms of how plant development and disease resistance are coordinately programed remain elusive. Here, we showed that the basic Helix-Loop-Helix (bHLH) transcription factor Cucumis sativus Irregular Vasculature Patterning (CsIVP) was highly expressed in cucumber vascular tissues. Knockdown of CsIVP caused severe vasculature disorganization and abnormal organ morphogenesis. CsIVP directly binds to vascular-related regulators YABBY5 (CsYAB5), BREVIPEDICELLUS (CsBP), and AUXIN/INDOLEACETIC ACIDS4 (CsAUX4) and promotes their expression. Knockdown of CsYAB5 resulted in similar phenotypes as CsIVP-RNA interference (RNAi) plants, including disturbed vascular configuration and abnormal organ morphology. Meanwhile, CsIVP-RNAi plants were more resistant to downy mildew and accumulated more salicylic acid (SA). CsIVP physically interacts with NIM1-INTERACTING1 (CsNIMIN1), a negative regulator in the SA signaling pathway. Thus, CsIVP is a novel vasculature regulator functioning in CsYAB5-mediated organ morphogenesis and SA-mediated downy mildew resistance in cucumber.

Lettuce (Lactuca sativa L.) is a very important leafy vegetable in China and is commonly grown using furrow irrigation. In order to improve production efficiency, greenhouse experiments were conducted at Experimental Station, China Agricultural University, Beijing, China using furrow irrigation (FI), micro-sprinkler irrigation (MS), plastic film mulching irrigation (PF) and a combined plastic film mulching-micro-sprinkler irrigation system (PF+MS) to study their effects on soil physical characteristics, water distribution, root morpho-physiological traits, nutrition absorption, lettuce yield and water use efficiency for a spring crop and autumn crop in 2015 (Fig 1). Root length, root surface area, and root density were significantly higher under PF and PF+MS than under FI. Moreover, these traits were higher under MS than under FI but these differences were not significant. The soluble protein, soluble sugar, and Vitamin C content of lettuce decreased in the order PF+MS > PF > MS > FI in both crops. In the spring crop, the biological yield of MS, PF, and PF+MS was 7.22%、36.77%、43.20% higher than FI, respectively. In the spring crop, biological water use efficiency (BWUE) of FI, MS, PF and PF+MS was 20.93, 25.24, 36.81 and 38.54 kg m-3, respectively. The BWUE of MS, PF, and PF+MS was 20.59%, 75.87% and 84.14% higher than FI. Economic water use efficiency (EWUE) of FI, MS, PF and PF+MS was 17.06, 21.31, 31.11 and 32.31 kg m-3, respectively. The EWUE of MS, PF, and PF+MS was 24.91%, 82.36% and 89.39% higher than FI, respectively. The autumn crop achieved a higher WUE than the spring crop. The results suggested that the combined plastic film mulching-micro-sprinkler irrigation was the most suitable irrigation approach for increasing lettuce yield.

Lettuce (Lactuca sativa L.) is an important leafy vegetable consumed worldwide. Heat-induced bolting and flowering greatly limit lettuce production during the summer. Additionally, MADS-box transcription factors are important for various aspects of plant development and architecture (e.g., flowering and floral patterning). However, there has been no comprehensive study of lettuce MADS-box family genes. In this study, we identified 82 MADS-box family genes in lettuce, including 23 type I genes and 59 type II genes. Transcriptome profiling revealed that LsMADS gene expression patterns differ among the various floral stages and organs. Moreover, heat-responsive cis-elements were detected in the promoter regions of many LsMADS genes. An in situ hybridization assay of 10 homologs of flower-patterning genes and a yeast two-hybrid assay of the encoded proteins revealed that the ABC model is conserved in lettuce. Specifically, the APETALA1 (AP1) homolog in lettuce, LsMADS55, is responsive to heat and is specifically expressed in the inflorescence meristem and pappus bristles. The overexpression of LsMADS55 results in early flowering in Arabidopsis thaliana. Furthermore, we observed that the heat shock factor LsHSFB2A-1 can bind to the LsMADS55 promoter in lettuce. Therefore, a model was proposed for the LsMADS-regulated floral organ specification and heat-induced flowering in lettuce.

Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable that is consumed during its vegetative growth. The transition from vegetative to reproductive growth is induced by high temperature, which has significant economic effect on lettuce production. However, the progression of floral transition and the molecular regulation of bolting are largely unknown. Here we morphologically characterized the inflorescence development and functionally analyzed the FLOWERING LOCUS T (LsFT) gene during bolting regulation in lettuce. We described the eight developmental stages during floral transition process. The expression of LsFT was negatively correlated with bolting in different lettuce varieties, and was promoted by heat treatment. Overexpression of LsFT could recover the late-flowering phenotype of ft-2 mutant. Knockdown of LsFT by RNA interference dramatically delayed bolting in lettuce, and failed to respond to high temperature. Therefore, this study dissects the process of inflorescence development and characterizes the role of LsFT in bolting regulation in lettuce.

Lettuce (Lactuca sativa L.) is one of the most economically important vegetables. The floral transition in lettuce is accelerated under high temperatures, which can significantly decrease yields. However, the molecular mechanism underlying the floral transition in lettuce is poorly known. Using laser capture microdissection coupled with RNA sequencing, we isolated shoot apical meristem cells from the bolting-sensitive lettuce line S39 at four critical stages of development. Subsequently, we screened specifically for the flowering-related gene LsSOC1 during the floral transition through comparative transcriptomic analysis. Molecular biology, developmental biology, and biochemical tools were combined to investigate the biological function of LsSOC1 in lettuce. LsSOC1 knockdown by RNA interference resulted in a significant delay in the timing of bolting and insensitivity to high temperature, which indicated that LsSOC1 functions as an activator during heat-promoted bolting in lettuce. We determined that two heat shock transcription factors, HsfA1e and HsfA4c, bound to the promoter of LsSOC1 to confirm that LsSOC1 played an important role in heat-promoted bolting. This study indicates that LsSOC1 plays a crucial role in the heat-promoted bolting process in lettuce. Further investigation of LsSOC1 may be useful for clarification of the bolting mechanism in lettuce.

Anther and ovule genesis preconditions crop fertilization and fruit production; however, coordinative regulation of anther and ovule development and underlying molecular pathways remain largely elusive. Here, we found that SPOROCYTELESS (SPL)/NOZZLE (NZZ) expression was nearly abolished in a Cucumis sativus (cucumber) mutant with severely defective anther and ovule development. CsSPL was expressed specifically in the developing anthers and ovules. Knock-down of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development. Importantly, CsSPL directly interacted with CsWUS (WUSCHEL) in the nucellus and YABBY family genes in integuments, and positively regulated CsWUS expression, meanwhile the HD-ZIP III gene CsPHB (PHABULOSA), expressed specifically in the nucellus, promoted CsSPL expression by binding to the CsSPL promoter. Thus, CsSPL acts as an adaptor to link CsPHB and CsWUS functioning, underpinning a previously unidentified regulatory pathway orchestrating sex organ development in planta. In addition, auxin accumulation was reduced in the reproductive organs of CsSPL knock-down plants. Biochemical analyses further showed that CsSPL stimulated the expression of AUXIN RESPONSE FACTOR 3 (CsARF3), and was positively regulated by CsARF13 during reproductive organ development, indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.

Bolting in lettuce is promoted by high temperature and bolting resistance is of great economic importance for lettuce production. But how bolting is regulated at the molecular level remains elusive. Here, a bolting resistant line S24 and a bolting sensitive line S39 were selected for morphological, physiological, transcriptomic and proteomic comparisons. A total of 12204 genes were differentially expressed in S39 vs S24. Line S39 was featured with larger leaves, higher levels of chlorophyll, soluble sugar, anthocyanin and auxin, consistent with its up-regulation of genes implicated in photosynthesis, oxidation-reduction and auxin actions. Proteomic analysis identified 30 differentially accumulated proteins in lines S39 and S24 upon heat treatment, and 19 out of the 30 genes showed differential expression in the RNA-Seq data. Exogenous gibberellins (GA) treatment promoted bolting in both S39 and S24, while 12 flowering promoting MADS-box genes were specifically induced in line S39, suggesting that although GA regulates bolting in lettuce, it may be the MADS-box genes, not GA, that plays a major role in differing the bolting resistance between these two lettuce lines.

The phytohormone auxin is essential for plant growth and development, and YUCCA (YUC) proteins catalyze a rate-limiting step for endogenous auxin biosynthesis. Despite YUC family genes have been isolated from several species, systematic expression analyses of YUCs in response to abiotic stress are lacking, and little is known about the function of YUC homologs in agricultural crops. Cucumber (Cucumis sativus L.) is a world cultivated vegetable crop with great economical and nutritional value. In this study, we isolated 10 YUC family genes (CsYUCs) from cucumber and explored their expression pattern under four types of stress treatments. Our data showed that CsYUC8 and CsYUC9 were specifically upregulated to elevate the auxin level under high temperature. CsYUC10b was dramatically increased but CsYUC4 was repressed in response to low temperature. CsYUC10a and CsYUC11 act against the upregulation of CsYUC10b under salinity stress, suggesting that distinct YUC members participate in different stress response, and may even antagonize each other to maintain the proper auxin levels in cucumber. Further, CsYUC11 was specifically expressed in the male flower in cucumber, and enhanced tolerance to salinity stress and regulated pedicel and stamen development through auxin biosynthesis in Arabidopsis.

Shoot organ primordia are initiated from the shoot apical meristem and develop into leaves during the vegetative stage, and into flowers during the reproductive phase. Between the meristem and the newly formed organ primordia, a boundary with specialized cells is formed that separates meristematic activity from determinate organ growth. Despite interactions that have been found between boundary regulators with genes controlling meristem maintenance or primordial development, most boundary studies were performed during embryogenesis or vegetative growth, hence little is known about whether and how boundaries communicate with meristem and organ primordia during the reproductive stage. We combined genetic, molecular and biochemical tools to explore interactions between the boundary gene HANABA TARANU (HAN) and two meristem regulators BREVIPEDICELLUS (BP) and PINHEAD (PNH), and three primordia-specific genes PETAL LOSS (PTL), JAGGED (JAG) and BLADE-ON-PETIOLE (BOP) during flower development. We demonstrated the key role of HAN in determining petal number, as part of a set of complex genetic interactions. HAN and PNH transcriptionally promote each other, and biochemically interact to regulate meristem organization. HAN physically interacts with JAG, and directly stimulates the expression of JAG and BOP2 to regulate floral organ development. Further, HAN directly binds to the promoter and intron of CYTOKININ OXIDASE 3 (CKX3) to modulate cytokinin homeostasis in the boundary. Our data suggest that boundary-expressing HAN communicates with the meristem through the PNH, regulates floral organ development via JAG and BOP2, and maintains boundary morphology through CKX3 during flower development in Arabidopsis.