The AGAMOUS-LIKE 16-GENERAL REGULATORY FACTOR 1 module regulates axillary bud outgrowth via catabolism of abscisic acid in cucumber.

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.

Identification and functional characterization of conserved cis-regulatory elements responsible for early fruit development in cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved noncoding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865 to 43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2, and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber.

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 CsCRCA is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRCA exhibited a 34∼39% reduction in fruit length. Introducing CsCRCG into this NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRCG expression was positively correlated with fruit length. Further, CsCRCG, but not CsCRCA, 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 CsCRCG 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.

Cucumber STACHYOSE SYNTHASE is regulated by its cis-antisense RNA asCsSTS to balance source-sink carbon partitioning.

Photosynthate partitioning between source and sink is a key determinant of crop yield. In contrast to sucrose-transporting plants, cucumber (Cucumis sativus) plants mainly transport stachyose and stachyose synthase (CsSTS) synthesizes stachyose in the vasculature for loading. Therefore, CsSTS is considered a key regulator of carbon partitioning. We found that CsSTS expression and CsSTS enzyme activity were upregulated in the vasculature and downregulated in mesophyll tissues at fruiting. In situ hybridization and tissue enrichment experiments revealed that a cis-natural antisense noncoding transcript of CsSTS, named asCsSTS, is mainly expressed in mesophyll tissues. In vitro overexpression (OE), RNA interference (RNAi), and dual luciferase reporter experiments indicated that CsSTSs are negatively regulated by asCsSTS. Fluorescence in situ hybridization revealed that asCsSTS transcript localized in leaf cytoplasm, indicating that the regulation of CsSTS by asCsSTS is a posttranscriptional process. Further investigation revealed that this regulation occurred by reducing CsSTS transcript stability through a DICER-like protein-mediated pathway. Chemically induced OE and RNAi of asCsSTS led to promotion or inhibition, respectively, of assimilate export from leaves and altered fruit growth rates. Our results suggest that the regulation of CsSTSs between the mesophyll and vasculature reduces sugar storage in mesophyll tissue and promotes assimilate export from the leaf when the plant carries fruit.

Control of the mitotic cleavage plane by local epithelial topology.

For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals.

CsPM5.2, a phosphate transporter protein-like gene, promotes powdery mildew resistance in cucumber.

Powdery mildew (PM) is one of the most serious fungal diseases affecting cucumbers (Cucumis sativus L.). The mechanism of PM resistance in cucumber is intricate and remains fragmentary as it is controlled by several genes. In this study, we detected the major-effect Quantitative Trait Locus (QTL), PM5.2, involved in PM resistance by QTL mapping. Through fine mapping, the dominant PM resistance gene, CsPM5.2, was cloned and its function was confirmed by transgenic complementation and natural variation identification. In cultivar 9930, a dysfunctional CsPM5.2 mutant resulted from a single nucleotide polymorphism in the coding region and endowed susceptibility to PM. CsPM5.2 encodes a phosphate transporter-like protein PHO1; H3. The expression of CsPM5.2 is ubiquitous and induced by the PM pathogen. In cucumber, both CsPM5.2 and Cspm5.1 (Csmlo1) are required for PM resistance. Transcriptome analysis suggested that the salicylic acid (SA) pathway may play an important role in CsPM5.2-mediated PM resistance. Our findings help parse the mechanisms of PM resistance and provide strategies for breeding PM-resistant cucumber cultivars.

Cucumber (Cucumis sativus L.) translationally controlled tumor protein interacts with CsRab11A and promotes activation of target of rapamycin in response to Podosphaera xanthii.

The target of rapamycin (TOR) kinase serves as a central regulator that integrates nutrient and energy signals to orchestrate cellular and organismal physiology in both animals and plants. Despite significant advancements having been made in understanding the molecular and cellular functions of plant TOR kinases, the upstream regulators that modulate TOR activity are not yet fully elucidated. In animals, the translationally controlled tumor protein (TCTP) is recognized as a key player in TOR signaling. This study reveals that two TCTP isoforms from Cucumis sativus, when introduced into Arabidopsis, are instrumental in balancing growth and defense mechanisms against the fungal pathogen Golovinomyces cichoracearum. We hypothesize that plant TCTPs act as upstream regulators of TOR in response to powdery mildew caused by Podosphaera xanthii in Cucumis. Our research further uncovers a stable interaction between CsTCTP and a small GTPase, CsRab11A. Transient transformation assays indicate that CsRab11A is involved in the defense against P. xanthii and promotes the activation of TOR signaling through CsTCTP. Moreover, our findings demonstrate that the critical role of TOR in plant disease resistance is contingent upon its regulated activity; pretreatment with a TOR inhibitor (AZD-8055) enhances cucumber plant resistance to P. xanthii, while pretreatment with a TOR activator (MHY-1485) increases susceptibility. These results suggest a sophisticated adaptive response mechanism in which upstream regulators, CsTCTP and CsRab11A, coordinate to modulate TOR function in response to P. xanthii, highlighting a novel aspect of plant-pathogen interactions.

Deep learning-based association analysis of root image data and cucumber yield.

The root system is important for the absorption of water and nutrients by plants. Cultivating and selecting a root system architecture (RSA) with good adaptability and ultrahigh productivity have become the primary goals of agricultural improvement. Exploring the correlation between the RSA and crop yield is important for cultivating crop varieties with high-stress resistance and productivity. In this study, 277 cucumber varieties were collected for root system image analysis and yield using germination plates and greenhouse cultivation. Deep learning tools were used to train ResNet50 and U-Net models for image classification and segmentation of seedlings and to perform quality inspection and productivity prediction of cucumber seedling root system images. The results showed that U-Net can automatically extract cucumber root systems with high quality (F1_score ≥ 0.95), and the trained ResNet50 can predict cucumber yield grade through seedling root system image, with the highest F1_score reaching 0.86 using 10-day-old seedlings. The root angle had the strongest correlation with yield, and the shallow- and steep-angle frequencies had significant positive and negative correlations with yield, respectively. RSA and nutrient absorption jointly affected the production capacity of cucumber plants. The germination plate planting method and automated root system segmentation model used in this study are convenient for high-throughput phenotypic (HTP) research on root systems. Moreover, using seedling root system images to predict yield grade provides a new method for rapidly breeding high-yield RSA in crops such as cucumbers.

ELONGATED HYPOTCOTYL5 and SPINE BASE SIZE1 together mediate light-regulated spine expansion in cucumber.

Plant trichome development is influenced by diverse developmental and environmental signals, but the molecular mechanisms involved are not well understood in most plant species. Fruit spines (trichomes) are an important trait in cucumber (Cucumis sativus L.), as they affect both fruit smoothness and commercial quality. Spine Base Size1 (CsSBS1) has been identified as essential for regulating fruit spine size in cucumber. Here, we discovered that CsSBS1 controls a season-dependent phenotype of spine base size in wild-type plants. Decreased light intensity led to reduced expression of CsSBS1 and smaller spine base size in wild-type plants, but not in the mutants with CsSBS1 deletion. Additionally, knockout of CsSBS1 resulted in smaller fruit spine base size and eliminated the light-induced expansion of spines. Overexpression of CsSBS1 increased spine base size and rescued the decrease in spine base size under low light conditions. Further analysis revealed that ELONGATED HYPOTCOTYL5 (HY5), a major transcription factor involved in light signaling pathways, directly binds to the promoter of CsSBS1 and activates its expression. Knockout of CsHY5 led to smaller fruit spine base size and abolished the light-induced expansion of spines. Taken together, our study findings have clarified a CsHY5-CsSBS1 regulatory module that mediates light-regulated spine expansion in cucumber. This finding offers a strategy for cucumber breeders to develop fruit with stable appearance quality under changing light conditions.

Multiplex gene editing reveals cucumber MILDEW RESISTANCE LOCUS O family roles in powdery mildew resistance.

Powdery mildew (PM) is one of the most widespread and prevalent diseases that affects a wide range of crops. In cucumber (Cucumis sativus L.), previous forward genetic studies have identified MILDEW RESISTANCE LOCUS O 8 (CsMLO8) as necessary but alone insufficient for cucumber PM resistance (PMR) and suggested the involvement of other members of the CsMLO family. However, the function of other CsMLO family members in cucumber remains largely unknown. Here, we developed a highly efficient multiplex gene editing system in cucumber to generate a series of Csmlo mutants from all the 13 family members. Systematic analysis of these mutants revealed growth effects of these CsMLO family members on development and PMR. Importantly, we obtained the Csmlo1/8/11 triple mutant with complete resistance to PM. Transcriptome and proteome analysis of PM-resistant Csmlo mutants suggested that the kinesin-like calmodulin-binding protein (KCBP)-interacting Ca2+-binding protein (CsKIC), calmodulin-like protein 28 (CsCML28), and Ca2+-dependent protein kinase 11 (CsCPK11)-mediated calcium signaling pathway is involved in PMR. CsMLO8 interacted directly with CsKIC, and the simultaneous silencing of both genes resulted in a phenotype that resembled the silencing of CsKIC alone. Silencing CsCML28 and CsCPK11 increased susceptibility to PM, whereas overexpressing CsCPK11 through genetic transformation enhanced cucumber's PMR, demonstrating their positive regulatory roles in PMR. Given the importance of PMR for cucurbit crops, this research provides unprecedented insights into the function of the proteins encoded by the CsMLO gene family as well as the plant defense response to PM pathogen.

Transcription factor CsMYB36 regulates fruit neck length via mediating cell expansion in cucumber.

The fruit neck is an important agronomic trait of cucumber (Cucumis sativus). However, the underlying genes and regulatory mechanisms involved in fruit neck development are poorly understood. We previously identified a cucumber yellow-green peel (ygp) mutant, whose causal gene is MYB DOMAIN PROTEIN 36 (CsMYB36). This study showed that the ygp mutant exhibited a shortened fruit neck and repressed cell expansion in the fruit neck. Further functional analysis showed that CsMYB36 was also a target gene, and its expression was enriched in the fruit neck. Overexpression of CsMYB36 in the ygp mutant rescued shortened fruit necks. Furthermore, transcriptome analysis and reverse transcription quantitative PCR (RT-qPCR) assays revealed that CsMYB36 positively regulates the expression of an expansin-like A3 (CsEXLA3) in the fruit neck, which is essential for cell expansion. Yeast 1-hybrid and dual-luciferase assays revealed that CsMYB36 regulates fruit neck elongation by directly binding to the promoter of CsEXLA3. Collectively, these findings demonstrate that CsMYB36 is an important gene in the regulation of fruit neck length in cucumber plants.

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

Cell wall invertase 3 plays critical roles in providing sugars during pollination and fertilization in cucumber.

In plants, pollen-pistil interactions during pollination and fertilization mediate pollen hydration and germination, pollen tube growth, and seed set and development. Cell wall invertases (CWINs) help provide the carbohydrates for pollen development; however, their roles in pollination and fertilization have not been well established. In cucumber (Cucumis sativus), CsCWIN3 showed the highest expression in flowers, and we further examined CsCWIN3 for functions during pollination to seed set. Both CsCWIN3 transcript and CsCWIN3 protein exhibited similar expression patterns in the sepals, petals, stamen filaments, anther tapetum, and pollen of male flowers, as well as in the stigma, style, transmitting tract, and ovule funiculus of female flowers. Notably, repression of CsCWIN3 in cucumber did not affect the formation of parthenocarpic fruit but resulted in an arrested growth of stigma integuments in female flowers and a partially delayed dehiscence of anthers with decreased pollen viability in male flowers. Consequently, the pollen tube grew poorly in the gynoecia after pollination. In addition, CsCWIN3-RNA interference plants also showed affected seed development. Considering that sugar transporters could function in cucumber fecundity, we highlight the role of CsCWIN3 and a potential close collaboration between CWIN and sugar transporters in these processes. Overall, we used molecular and physiological analyses to determine the CsCWIN3-mediated metabolism during pollen formation, pollen tube growth, and plant fecundity. CsCWIN3 has essential roles from pollination and fertilization to seed set but not parthenocarpic fruit development in cucumber.

A nuclease-dead Cas9-derived tool represses target gene expression.

Manipulation of gene expression is central to understanding gene function, engineering cell behavior, and altering biological traits according to production demands. Nuclease-dead Cas9 (dCas9), a variant of active Cas9, offers a versatile platform for the precise control of genome function without DNA cleavage. Notably, however, an effective and universal dCas9-based transcriptional repression system remains unavailable in plants. The noncanonical histone acetyltransferase TENDRIL-LESS (CsTEN) is responsible for chromatin loosening and histone modification in cucumber (Cucumis sativus). In this study, we engineered a gene regulation tool by fusing TEN and its truncated proteins with dCas9. The full-length dCas9-TEN protein substantially repressed gene expression, with the N-terminal domain identified as the core repression domain. We subsequently validated the specificity and efficacy of this system through both transient infection and genetic transformation in cucumber and Arabidopsis (Arabidopsis thaliana). The electrophoretic mobility shift assay (EMSA) revealed the ability of the N-terminal domain of TEN to bind to chromatin, which may promote target binding of the dCas9 complex and enhance the transcriptional repression effect. Our tool enriches the arsenal of genetic regulation tools available for precision breeding in crops.

The CsHEC1-CsOVATE module contributes to fruit neck length variation via modulating auxin biosynthesis in cucumber.

Fruit neck is the proximal portion of the fruit with undesirable taste that has detrimental effects on fruit shape and commercial value in cucumber. Despite the dramatic variations in fruit neck length of cucumber germplasms, the genes and regulatory mechanisms underlying fruit neck elongation remain mysterious. In this study, we found that Cucumis sativus HECATE1 (CsHEC1) was highly expressed in fruit neck. Knockout of CsHEC1 resulted in shortened fruit neck and decreased auxin accumulation, whereas overexpression of CsHEC1 displayed the opposite effects, suggesting that CsHEC1 positively regulated fruit neck length by modulating local auxin level. Further analysis showed that CsHEC1 directly bound to the promoter of the auxin biosynthesis gene YUCCA4 (CsYUC4) and activated its expression. Enhanced expression of CsYUC4 resulted in elongated fruit neck and elevated auxin content. Moreover, knockout of CsOVATE resulted in longer fruit neck and higher auxin. Genetic and biochemical data showed that CsOVATE physically interacted with CsHEC1 to antagonize its function by attenuating the CsHEC1-mediated CsYUC4 transcriptional activation. In cucumber germplasms, the expression of CsHEC1 and CsYUC4 positively correlated with fruit neck length, while that of CsOVATE showed a negative correlation. Together, our results revealed a CsHEC1-CsOVATE regulatory module that confers fruit neck length variation via CsYUC4-mediated auxin biosynthesis in cucumber.

A CsEIL3-CsARN6.1 module promotes waterlogging-triggered adventitious root formation in cucumber by activating the expression of CsPrx5.

The formation of adventitious roots (ARs) derived from hypocotyl is the most important morphological adaptation to waterlogging stress in Cucumis sativus (cucumber). Our previous study showed that cucumbers with the gene CsARN6.1, encoding an AAA ATPase domain-containing protein, were more tolerant to waterlogging through increased AR formation. However, the apparent function of CsARN6.1 remained unknown. Here, we showed that the CsARN6.1 signal was predominantly observed throughout the cambium of hypocotyls, where de novo AR primordia are formed upon waterlogging treatment. The silencing of CsARN6.1 expression by virus-induced gene silencing and CRISPR/Cas9 technologies adversely affects the formation of ARs under conditions of waterlogging. Waterlogging treatment significantly induced ethylene production, thus upregulating CsEIL3 expression, which encodes a putative transcription factor involved in ethylene signaling. Furthermore, yeast one-hybrid, electrophoretic mobility assay and transient expression analyses showed that CsEIL3 binds directly to the CsARN6.1 promoter to initiate its expression. CsARN6.1 was found to interact with CsPrx5, a waterlogging-responsive class-III peroxidase that enhanced H2 O2 production and increased AR formation. These data provide insights into understanding the molecular mechanisms of AAA ATPase domain-containing protein and uncover a molecular mechanism that links ethylene signaling with the formation of ARs triggered by waterlogging.

TARGET OF EAT3 (TOE3) specifically regulates fruit spine initiation in cucumber (Cucumis sativus L.).

The presence or absence of spines is an important economic trait of cucumber fruit. Spines are believed to be a type of specialized trichome on the fruit surface, and all the identified cucumber trichome-less mutants lack fruit spines. However, genes that specifically regulate fruit spine initiation remain to be identified. Here, we found that knocking out cucumber TARGET OF EAT3 homolog (CsTOE3), belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, affected flower development and, more interestingly, inhibited cucumber fruit spine initiation. On analyzing expression patterns by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization assay, CsTOE3 was found to be highly expressed in male and female flowers, and its mRNA accumulated in the tips of sepal and petal primordia and in the cells of fruit spines and peels. Biochemical analyses indicated that CsTOE3 directly interacts with GLABRA1 (CsGL1) and TRANSPARENT TESTA GLABRA1 (CsTTG1), which are positive regulators of trichome formation. In addition, RNA-seq showed that the transcription levels of eight ERFs were significantly upregulated in CsTOE3 knockout lines. Phytohormone content analysis also revealed a significant increase in the amount of ethylene released by CsTOE3 knockout line, and treatment with the ethylene synthesis inhibitor aminoethoxyvinyl-glycine partly restored the spineless phenotype. Our results suggest that CsTOE3 specifically regulates fruit spine initiation but does not affect the formation of trichomes on other organs in cucumber. Our findings may have a far-reaching significance for cucumber germplasm improvement and quality breeding using fruit spines as the target trait.

Comparative transcriptome analysis revealed that auxin and cell wall biosynthesis play important roles in the formation of hollow hearts in cucumber.

BACKGROUND: Hollow heart is a kind of physiological defect that seriously affects the yield, quality, and economic value of cucumber. However, the formation of hollow hearts may relate to multiple factors in cucumber, and it is necessary to conduct analysis. RESULTS: In this study, hollow and non-hollow fruits of cucumber K07 were used for comparative transcriptome sequencing and analysis. 253 differentially expressed genes and 139 transcription factors were identified as being associated with the formation of hollow hearts. Hormone (auxin) signaling and cell wall biosynthesis were mainly enriched in GO and KEGG pathways. Expression levels of key genes involved in indole-3-acetic acid biosynthesis in carpel were lower in the hollow fruits than non-hollow fruits, while there was no difference in the flesh. The concentration of indole-3-acetic also showed lower in the carpel than flesh. The biosynthetic pathway and content analysis of the main components of the cell wall found that lignin biosynthesis had obvious regularity with hollow heart, followed by hemicellulose and cellulose. Correlation analysis showed that there may be an interaction between auxin and cell wall biosynthesis, and they collectively participate in the formation of hollow hearts in cucumber. Among the differentially expressed transcription factors, MYB members were the most abundant, followed by NAC, ERF, and bHLH. CONCLUSIONS: The results and analyses showed that the low content of auxin in the carpel affected the activity of enzymes related to cell wall biosynthesis at the early stage of fruit development, resulting in incomplete development of carpel cells, thus forming a hollow heart in cucumber. Some transcription factors may play regulatory roles in this progress. The results may enrich the theory of the formation of hollow hearts and provide a basis for future research.

CsSHMT3 gene enhances the growth and development in cucumber seedlings under salt stress.

Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2O2), hydrogen peroxide (O2·-) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed. However, the content of chlorophyll and Pro, photosynthetic rate, and antioxidant enzyme activity of the silenced CsSHMT3 gene lines under salt stress were significantly reduced, while MDA, H2O2, O2·- content and relative conductivity showed higher level in the silenced CsSHMT3 gene lines. It was further found that the expression of stress-related genes SOD, CAT, SOS1, SOS2, NHX, and HKT was significantly up-regulated by overexpressing CsSHMT3 gene in cucumber seedlings; while stress-related gene expression showed significant decrease in silenced CsSHMT3 gene seedlings under salt stress. This suggests that overexpression of CsSHMT3 gene increased the salt tolerance of cucumber seedlings, while silencing of CsSHMT3 gene decreased the salt tolerance. In conclusion, CsSHMT3 gene might positively regulate salt stress tolerance in cucumber and be involved in regulating antioxidant activity, osmotic adjustment, and photosynthesis under salt stress. KEY MESSAGE: CsSHMT3 gene may positively regulate the expression of osmotic system, photosynthesis, antioxidant system and stress-related genes in cucumber.

CsHSFA1d Promotes Drought Stress Tolerance by Increasing the Content of Raffinose Family Oligosaccharides and Scavenging Accumulated Reactive Oxygen Species in Cucumber.

Drought is the most severe form of stress experienced by plants worldwide. Cucumber is a vegetable crop that requires a large amount of water throughout the growth period. In our previous study, we identified that overexpression of CsHSFA1d could improve cold tolerance and the content of endogenous jasmonic acid in cucumber seedlings. To explore the functional diversities of CsHSFA1d, we treat the transgenic plants under drought conditions. In this study, we found that the heat shock transcription factor HSFA1d (CsHSFA1d) could improve drought stress tolerance in cucumber. CsHSFA1d overexpression increased the expression levels of galactinol synthase (CsGolS3) and raffinose synthase (CsRS) genes, encoding the key enzymes for raffinose family oligosaccharide (RFO) biosynthesis. Furthermore, the lines overexpressing CsHSFA1d showed higher enzymatic activity of GolS and raffinose synthase to increase the content of RFO. Moreover, the CsHSFA1d-overexpression lines showed lower reactive oxygen species (ROS) accumulation and higher ROS-scavenging enzyme activity after drought treatment. The expressions of antioxidant genes CsPOD2, CsAPX1 and CsSOD1 were also upregulated in CsHSFA1d-overexpression lines. The expression levels of stress-responsive genes such as CsRD29A, CsLEA3 and CsP5CS1 were increased in CsHSFA1d-overexpression lines after drought treatment. We conclude that CsHSFA1d directly targets and regulates the expression of CsGolS3 and CsRS to promote the enzymatic activity and accumulation of RFO to increase the tolerance to drought stress. CsHSFA1d also improves ROS-scavenging enzyme activity and gene expression indirectly to reduce drought-induced ROS overaccumulation. This study therefore offers a new gene target to improve drought stress tolerance in cucumber and revealed the underlying mechanism by which CsHSFA1d functions in the drought stress by increasing the content of RFOs and scavenging the excessive accumulation of ROS.