Zhongyi Wang

Heat stress has become a primary environmental factor that severely threatens crop yield and quality. Despite many genes having been identified to participate in heat response, natural alleles of heat response genes that can be utilized in crop breeding are scarce, and the underlying mechanisms remain enigmatic. In this study, we identified a BEL1-like Homeodomain (BLH) transcription factor CsBLH9, through genome-wide association study (GWAS), and demonstrated its positive role in heat response. The natural variants of CsBLH9 exhibit different levels of heat tolerance and differential response to gibberellin (GA) treatment. Knockout of CsBLH9 resulted in sensitive heat response and reduced chlorophyll levels. CsBLH9 can directly bind to the promoters of chlorophyll catabolic genes stay-green (CsSGR) and pheophorbide a oxygenase (CsPAO) to inhibit their expression. Furthermore, we identified CsOVATE as an interacting partner of CsBLH9 that also functions as a positive regulator of heat tolerance. Csovate mutants showed phenotypes similar to Csblh9 mutants under heat stress, and CsOVATE enhanced the CsBLH9-mediated inhibition of CsSGR and CsPAO expression. Therefore, our findings suggested that CsBLH9-CsOVATE acts as a module that regulates heat tolerance via chlorophyll degradation in cucumber, and the natural alleles of CsBLH9 provide new targets for crop improvement under heat stress.

Hypocotyl length is a core indicator for seedling quality that directly affects vegetative growth and crop final yield. Despite intensive studies have been investigated the mutual promotion effect of auxin and brassinosteroids (BRs) in shaping hypocotyl elongation in Arabidopsis, the regulatory relationship between auxin and BR during hypocotyl development in horticultural crops remains largely unknown. In this study, we investigated the function of HECATE1 (CsHEC1) in cucumber seedlings and found that CsHEC1 positively regulates hypocotyl elongation. CsHEC1 can directly activate the expression of auxin biosynthesis YUCCA8 (CsYUC8), thereby promoting auxin biosynthesis. Exogenous application of the 1-Naphthaleneacetic acid (NAA) to Cshec1 mutants could mostly rescue the short-hypocotyl phenotype. Mutation of BRASSINOSTEROID INSENSITIVE 2 (BIN2), a negative regulator in the BR signaling pathway, resulted in long-hypocotyl phenotype upon 24-epibrassinolide (24-eBL) treatment in cucumber. However, the promoting effect of 24-eBL on hypocotyl elongation was abolished in Cshec1 mutants. Biochemical analyses demonstrated that CsBIN2 can interact with and phosphorylate CsHEC1, which consequently triggers the degradation of CsHEC1 and thus impairs the activation of CsYUC8. The hypocotyl length of Csbin2 Cshec1 double mutant resemble that of Cshec1 single mutant, indicating that CsBIN2 acts upstream of CsHEC1. In summary, our data suggested that CsHEC1 integrated BR signaling and auxin biosynthesis to regulate cucumber hypocotyl elongation, thus providing a valuable target for high-quality seedling breeding in cucumber.

HY5 is a key transcription factor integrating light and stress signals. However, its role in waterlogging adaptation remains unclear. This study unveils CaHY5 as a central hub integrating waterlogging signaling with auxin-mediated adventitious root formation in pepper. Wild-type and Cahy5 mutant pepper plants were subjected to waterlogging treatment. Subsequent statistical analysis of the number and temporal dynamics of adventitious root formation, combined with transcriptome data analysis, confirmed that CaHY5 acts as a negative regulator of waterlogging-induced adventitious root formation. Mechanistically, CaHY5 modulates auxin accumulation in the hypocotyl by directly repressing the expression of auxin transporters CaPIN1, while simultaneously suppressing auxin signaling by promoting the expression of CaIAA17. This dual regulatory action collectively attenuates the auxin response and inhibits adventitious root formation. In addition, CaHY5 can directly bind to the promoter regions of CaWOX5, CaLBD16, and CaSBRL, and inhibit their transcriptional activity, thereby suppressing adventitious root formation. However, the expression of CaHY5 is down-regulated under waterlogging stress, thereby alleviating its inhibitory effect on adventitious root formation. Our findings thus reveal a novel mechanism of waterlogging adaptation and identify CaHY5 as a key regulator coordinating root plasticity under waterlogging stress.

Sex determination underpins genesis of male and female flowers with particularly important implications in plant breeding. Auxin and ethylene regulate femaleness in cucurbits. In this work, we identified an auxin response factor 3 ( CsARF3 ) that plays an essential role in carpel development in cucumber. Deletion of CsARF3 resulted in an androecious phenotype with only male flowers, whereas overexpression of CsARF3 led to an increased number of female flowers. CsARF3 promotes femaleness by directly stimulating the expression of the meristem maintenance gene CsSTM and repressing the activity of the gynoecious gene CsWIP1 . Auxin and ethylene exhibit a reciprocal relationship during sex determination, in which ethylene promotes carpel formation through auxin at the early stage of flower development. The auxin signaling in carpels then enhances ethylene biosynthesis to inhibit stamen development.

Fruit shape is a key determinant of pepper (Capsicum annuum L.) fruit commercial value, and the shoulder is a crucial feature of the fruit's proximal end. Despite the fruit shoulder being an important parameter in morphological variation, the genes and regulatory mechanisms underlying fruit shoulder morphology remain elusive. Here, we identified a fruit shoulder (fs) mutant that primarily exhibits a shoulderless fruit phenotype, with an ∼40% reduction in fruit weight. Our data demonstrated that OVATE family protein 20 (CaOFP20) controls this phenotype. Silencing CaOFP20 in various fruit types consistently led to decreased fruit shoulder width, while fruit length was unaffected. CaOFP20 was mainly expressed in the pericarp, seeds, and placenta, where it regulates organ size by modulating cell division. Loss of CaOFP20 disrupted the expression of auxin pathway genes and the spatial distribution of auxin in fruits. Additionally, we demonstrated that CaOFP20 physically interacts with HECATE1 (CaHEC1), and silencing CaHEC1 in fs resulted in a wider fruit shoulder. Moreover, CaOFP20 and CaHEC1 play distinct yet complementary roles in regulating the expression of auxin pathway genes involved in fruit shoulder development, such as PIN-FORMED1 (CaPIN1), CaPIN3, and Indole-3-Acetic Acid Inducible (CaIAA9), thus finely controlling fruit shoulder development. Taken together, our findings establish CaOFP20 as a central regulator of fruit shoulder development and suggest its potential utility in breeding programs to enhance pepper fruit quality.

Chlorophylls and carotenoids are two pivotal photosynthetic pigments directly influencing the economic value of pepper (Capsicum annuum L.) fruits. However, the coordinated regulatory mechanisms governing the accumulation of both chlorophylls and carotenoids during pepper fruit development remain elusive. In this study, pepper B-box 10 (CaBBX10), a candidate hub transcription factor, was found to play dual roles in the early development of pepper fruit. CaBBX10 virus-induced gene silencing and overexpression experiments demonstrated that the encoded transcription factor promotes both chlorophyll and carotenoid accumulation in pepper fruit. Further comprehensive analyses showed that CaBBX10 directly binds to the promoter of magnesium chelatase subunit D subunit (CaCHLD) and phytoene synthase 1 (CaPSY1), thereby activating their expression in the chlorophyll and carotenoid biosynthesis pathways, respectively. Additionally, the photomorphogenic factor CaCOP1 was found to physically interact with CaBBX10 and lead to its degradation. Therefore, CaBBX10 may serve as a critical link connecting chlorophyll and carotenoid biosynthesis to light signaling. Altogether, our findings reveal a mechanism for the complex transcriptional regulation that simultaneously promotes chlorophyll and carotenoid accumulation in pepper fruit.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16–CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

In Arabidopsis, the photoreceptors phytochrome B- (PhyB) and UVB resistance 8- (UVR8) mediated light responses play a major role in regulating photomorphogenic hypocotyl growth, but how they crosstalk to coordinate this process is not well understood. Here we report map-based cloning and functional characterization of a UVB-insensitive, long-hypocotyl mutant, lh1, and a wild-type-like lh2 mutant in cucumber, Cucumis sativus, which encodes a defective CsPhyB and a key gibberellic acid (GA) biosynthesis enzyme CsGA20ox-2, respectively. The lh2 mutation was epistatic to lh1 and partly suppressed long-hypocotyl phenotype in the lhl1lh2 double mutant. We identified phytochrome interacting factor (PIF) CsPIF3 that played a critical role in integrating the red/far-red and UVB light responses for hypocotyl growth. We show that two modules, CsPhyB-CsPIF3-CsGA20ox-2 (GA oxidase2)-DELLA and CsPIF3-CsARF18 (auxin response factor 18) mediate CsPhyB-regulated hypocotyl elongation through GA and auxin pathways, respectively, in which CsPIF3 binds to the G-/E-box motifs in the promoters of CsGA20ox-2 and CsARF18 to regulate their expression. We also identified a new physical interaction between CsPIF3 and CsUVR8 mediating CsPhyB-dependent, UVB-induced hypocotyl growth inhibition. Our work suggests that hypocotyl growth in cucumber involves a complex interplay of multiple photoreceptor- and phytohormone-mediated signaling pathways that show both conservation with and divergence from those in Arabidopsis.

Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRC^A is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRC^A exhibited a 34∼39% reduction in fruit length. Introducing CsCRC^G into this NIL rescued the short-fruit phenotype, and knockdown of CsCRC^G resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRC^G expression was positively correlated with fruit length. Further, CsCRC^G, but not CsCRC^A, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRC^G positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.