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.

Real-time quantitative PCR (RT-qPCR) is a method with high sensitivity and convenience that has been extensively used to analyze the expression level of target genes. A reference gene with a highly stable expression is required to ensure the accuracy of experimental results. However, the report on appropriate reference genes in arrowheads (Sagittaria trifolia) is still limited. In this study, eight candidate reference genes (ACT5, UBQ, GAPDH, CYP, NAC, IDH, SLEEPER and PLA) were selected. The candidate genes were employed in a RT-qPCR assay in different tissues at different developmental stages of the same tissue (including corm, leaf and leafstalk) in arrowheads. Five statistical algorithms, GeNorm, NormFinder, BestKeeper, delta cycle threshold (ΔCt) and RefFinder, were used to evaluate the stability of these genes’ expressions in order to identify the appropriate reference genes. The results showed that UBQ was the optimum reference gene in leaf, leafstalk, root, stolon and corm, IDH exhibited the most stable expression during the expansion of corm, UBQ and PLA were the most stable reference genes in developmental stages of leaf and leafstalk, respectively. Finally, the reliability of reference genes was further confirmed by the normalization of PDS and EXP1 genes under different arrowhead tissues and developmental stages of corm, respectively. This study constitutes important guidance for the selection of reliable reference genes for analyzing the tissue- and developmental-stage-specific expression of genes in arrowheads.

The bZIP (basic leucine zipper) proteins play crucial roles in various biological functions. Nitrogen (N) is an essential element for plant growth, especially in cucumber (Cucumis sativus) due to its shallow roots. However, the regulation of bZIP genes in cucumber nitrogen metabolism has not been studied yet. In this study, we identified a total of 72 bZIP genes (CsbZIPs) in the cucumber genome that could be classified into 13 groups. These genes were unevenly distributed on seven chromosomes, and synteny analysis showed that the CsbZIP genes were expanded in a segmentally duplicating manner. Furthermore, our genome-wide expression analysis suggested that CsbZIP genes had different patterns and that five CsbZIP genes were regulated by nitrogen treatment in both leaves and roots. Consistent with CsNPF, CsbZIP55 and CsbZIP65 were regulated by nitrogen treatment in leaves and roots. Moreover, the subcellular localization showed that CsbZIP55 and CsbZIP65 were specifically located in the nucleus, and the transcriptional activation assay showed that CsbZIP55 and CsbZIP65 have transcriptional activation activity. Additionally, in the CsbZIP55 and CsbZIP65 overexpression plants, most nitrogen-regulated CsNPF genes were downregulated. Taken together, our comprehensive analysis of the bZIP gene family lays a foundation for understanding the molecular and physiological functions of CsbZIPs.

HD-Zip, a special class of transcription factors in high plants, has a role in plant development and responding to external environmental stress. Heat stress has always been an important factor affecting plant growth, quality, and yield. Carotenoid content is also an important factor affecting the color of the inner leaf blades of Chinese cabbage. In this study, the genomes of three Brassicaceae plants were selected: Chinese cabbage (Brassica rapa subsp. pekinensis), Brassica oleracea, and Brassica napus. We identified 93, 96, and 184 HD-Zip genes in the B. rapa, B. oleracea, and B. napus, respectively. The HD-Zip gene family was classified into four subfamilies based on phylogeny: I, II, III, and IV;. The results of cis-acting element analysis suggested that HD-Zip family genes may participate in various biological processes, such as pigment synthesis, cell cycle regulation, defense stress response, etc. Conserved motifs prediction revealed that three motifs exist among the four HD-Zip gene families and that different motifs exhibit significant effects on the structural differences in HD-Zips. Synteny, Ks, and 4DTv results displayed that genome-wide triplication events act in HD-Zip gene family expansion. Transcriptome data showed that 18 genes responded (>1.5-fold change) to heat stress in Chinese cabbage, and 14 of 18 genes were from the HD-Zip I subfamily. Three genes had up-regulation, and eight genes had down-regulation in high-carotenoid-content Chinese cabbage. The BraA09g011460.3C expression level was up-regulated after heat stress treatment and significantly reduced in varieties with high carotenoid content, indicating its potential for heat stress tolerance and carotenoid content regulation. This study provided important gene resources for the subsequent breeding of Chinese cabbage.

The optimization of the sink-source relationship is of great importance for crop yield regulation. Cucumber is a typical raffinose family oligosaccharide (RFO)-transporting crop. DNA methylation is a common epigenetic modification in plants, but its role in sink-source regulation has not been demonstrated in RFO-translocating species. Here, whole-genome bisulfite sequencing (WGBS-seq) was conducted to compare the nonfruiting-node leaves (NFNLs) and leaves of fruit setting (FNLs) at the 12th node by removing all female flowers in other nodes of the two treatments. We found considerable differentially methylated genes enriched in photosynthesis and carbohydrate metabolic processes. Comparative transcriptome analysis between FNLs and NFNLs indicated that many differentially expressed genes (DEGs) with differentially methylated regions were involved in auxin, ethylene and brassinolide metabolism; sucrose metabolism; and RFO synthesis pathways related to sink-source regulation. Moreover, DNA methylation levels of six sink-source-related genes in the pathways mentioned above decreased in leaves after 5-aza-dC-2′-deoxycytidine (5-Aza-dC, a DNA methyltransferase inhibitor) treatment on FNLs, and stachyose synthase (CsSTS) gene expression, enzyme activity and stachyose content in RFO synthesis pathway were upregulated, thereby increasing fruit length and dry weight. Taken together, our findings proposed an up-to-date inference for the potential role of DNA methylation in the sink-source relationship, which will provide important references for further exploring the molecular mechanism of DNA methylation in improving the yield of RFO transport plants.

Pumpkin (Cucurbita maxima) is an important vegetable crop of the Cucurbitaceae plant family. The fruits of pumpkin are often used as directly edible food or raw material for a number of processed foods. In nature, mature pumpkin fruits differ in size, shape, and color. The Atlantic Giant (AG) cultivar has the world’s largest fruits and is described as the giant pumpkin. AG is well-known for its large and bright-colored fruits with high ornamental and economic value. At present, there are insufficient studies that have focused on the formation factors of the AG cultivar. To address these knowledge gaps, we performed comparative transcriptome, proteome, and metabolome analysis of fruits from the AG cultivar and a pumpkin with relatively small fruit (Hubbard). The results indicate that up-regulation of gene-encoded expansins contributed to fruit cell expansion, and the increased presence of photoassimilates (stachyose and D-glucose) and jasmonic acid (JA) accumulation worked together in terms of the formation of large fruit in the AG cultivar. Notably, perhaps due to the rapid transport of photoassimilates, abundant stachyose that was not converted into glucose in time was detected in giant pumpkin fruits, implying that a unique mode of assimilate unloading is in existence in the AG cultivar. The potential molecular regulatory network of photoassimilate metabolism closely related to pumpkin fruit expansion was also investigated, finding that three MYB transcription factors, namely CmaCh02G015900, CmaCh01G018100, and CmaCh06G011110, may be involved in metabolic regulation. In addition, neoxanthin (a type of carotenoid) exhibited decreased accumulation that was attributed to the down-regulation of carotenoid biosynthesis genes in AG fruits, which may lead to pigmentation differences between the two pumpkin cultivars. Our current work will provide new insights into the potential formation factors of giant pumpkins for further systematic elucidation.