A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Size is the most important quality attribute of giant pumpkin fruit. Different concentrations and application frequencies of α-naphthaleneacetic acid (NAA) and 24-epibrassinolide (EBR) were sprayed on the leaves and fruits of giant pumpkin at different growth stages to determine their effects and the mechanism responsible for fruit size increase. NAA+EBR application improved source strength, and further analysis indicated that NAA+EBR markedly boosted net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and the expression level and activity of galactitol synthetase (GolS), raffinose synthetase (RS), and stachyose synthetase (STS), resulting in an increase in the synthesis of photoassimilate, especially stachyose. Concomitantly, NAA+EBR spray increased stachyose and sucrose contents throughout pumpkin fruit growth and the concentrations of glucose and fructose at 0 and 20 days post-anthesis (DPA) in peduncle phloem sap, implying that such treatment improved the efficiency of assimilate transport from the peduncle to the fruit. Furthermore, it improved the expression and activity of alkaline α-galactosidase (AGA), facilitating assimilate unloading, providing carbon skeletons and energy for fruit growth, and increasing fruit weight by more than 44.1%. Therefore, exogenous NAA and EBR increased source capacity, transportation efficiency, and sink strength, overall promoting the synthesis and distribution of photoassimilate, ultimately increasing fruit size.

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.

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) proteins perform an essential role in regulating plant nitrate absorption and distribution and in improving plant nitrogen use efficiency. In this study, cucumber (Cucumis sativus L.) NPF genes were comprehensively analyzed at the whole genome level, and 54 NPF genes were found to be unevenly distributed on seven chromosomes in the cucumber genome. The phylogenetic analysis showed that these genes could be divided into eight subfamilies. We renamed all CsNPF genes according to the international nomenclature, based on their homology with AtNPF genes. By surveying the expression profiles of CsNPF genes in various tissues, we found that CsNPF6.4 was specifically expressed in roots, indicating that CsNPF6.4 may play a role in N absorption; CsNPF6.3 was highly expressed in petioles, which may be related to NO₃⁻ storage in petioles; and CsNPF2.8 was highly expressed in fruits, which may promote NO₃⁻ transport to the embryos. We further examined their expression patterns under different abiotic stress and nitrogen conditions, and found that CsNPF7.2 and CsNPF7.3 responded to salt, cold, and low nitrogen stress. Taken together, our study lays a foundation for further exploration of the molecular and physiological functions of cucumber nitrate transporters.