Xiaolei Sui

SUMMARY: Male sterility is a vital trait for hybrid seed production. However, the synergistic coordination between sugar metabolism and reactive oxygen species (ROS) during pollen development remains poorly understood. Here, we identified a plastid‐localized hexokinase, CsHXK3, that participates in both processes in cucumber ( Cucumis sativus L.). CsHXK3 is localized to the tapetum, microspores, and vascular tissues of the anther. The significant downregulation of CsHXK3 expression in cell wall invertase 3 ‐silenced ( CsCWIN3 ‐RNAi) lines supports its essential role in downstream hexose utilization. Biochemical analysis confirmed that CsHXK3 is a glucose‐preferring enzyme that is essential for hexose phosphorylation. The CRISPR/Cas9‐mediated knockout of CsHXK3 resulted in severe male sterility, characterized by defective tapetal programmed cell death, collapsed pollen grains, and severely impaired pollen germination. Loss of CsHXK3 led to reduced accumulation of sugars (glucose, fructose, and sucrose) and starch in the anthers, accompanied by substantially downregulated expression of sugar transporter genes, including Sugars Will Eventually be Exported Transporters ( CsSWEETs ) and Sugar Transport Protein 13 ( CsSTP13 ). CsHXK3 deficiency disrupted ROS homeostasis by reducing hydrogen peroxide (H ₂ O ₂ ) levels, which was accompanied by the downregulated expression of the ROS‐generating gene Respiratory Burst Oxidase Homolog B ( CsRBOHB ) and upregulated expression of genes encoding ROS‐scavenging peroxidases. Our findings indicate that CsHXK3 may coordinate carbohydrate metabolism with ROS homeostasis during pollen development, offering insights into the metabolic regulation of male reproductive success in plants.

Lateral root (LR) branching is a vital adaptive strategy for plants to optimize nutrient acquisition, particularly under phosphorus deficiency. While auxin triggers LR formation, the molecular link between phosphate (Pi) homeostasis and the developmental competence of xylem-pole-pericycle (XPP) cells in roots remains elusive. In this study, we identified CsSPX2 from the SPX (SYG1/Pho81/XPR1) family as a critical phosphate sensor in cucumber (Cucumis sativus L.) that is strongly induced by Pi starvation. CRISPR/Cas9-generated CsSPX2 knockout lines displayed reduced lateral root density, shorter lateral roots and abnormal root meristems. The loss of CsSPX2 resulted in phosphate overaccumulation and excessive lignin deposition under Pi-sufficient conditions. Concomitantly, we observed abnormal thickening of the XPP cell walls and subsequently impaired auxin responsiveness at LR initiation sites, as evidenced by diminished DR5::GUS activity. Notably, under Pi-deficient conditions, spx2 mutants exhibited obviously decreased lignin content and significantly reduced thickness of XPP cell wall compared to Pi-sufficient conditions. In addition, the LR developmental defects in spx2 mutants were partially rescued by exogenous application of either a lignin biosynthesis inhibitor or synthetic auxin. Furthermore, CsSPX2 appeared to be important for maintaining the stem cell niche organization and meristematic activity in root tips, as its loss altered quiescent center and columella stem cell behavior and impaired root cap differentiation. Collectively, our findings support a model where CsSPX2 might link phosphate homeostasis to lateral root development through its involvement in XPP cell wall properties and auxin response. This study deepens our understanding of plant nutrition stress responses and provides a key genetic target for developing phosphate-efficient crops.

In the intricate landscape of plant−pathogen interactions, amino acids and their dedicated transporters emerge as pivotal players underpinning immune signalling and metabolic reprogramming. Amino acid metabolism serves as a linchpin in orchestrating systemic defence responses, with transporter‐mediated amino acid homoeostasis intricately intertwined with immune pathways. This review synthesizes the dual roles of amino acids, including glutamate, proline, γ‐aminobutyric acid, β‐aminobutyric acid and pipecolic acid, as metabolic intermediates and signalling molecules that modulate defence responses. Complementing this metabolic framework, amino acid transporters, including LHT1 and members of the AAP and UMAMIT family, participate in plant defence against pathogens or provide nutrients to pathogens by regulating the transmembrane transport of amino acids. Their disease resistance or susceptibility functions are closely related to plant tissue‐specificity and substrate‐specificity. Additionally, this review explores the potential coordinated regulation between amino acid and sugar transporters in the context of plant–pathogen interactions. Looking ahead, future research should focus on resolving transporter mechanisms in resistance, dissecting regulatory hubs linking metabolism and transport, mapping nutrient fluxes at the host−pathogen interface and exploring the subcellular localization and transport direction of transporters to inform precision crop protection strategies.

Seed size and weight are pivotal agronomic traits that link plant sexual reproduction to subsequent seedling establishment. These 2 seed characteristics are affected by embryo development and sugar filling. However, the molecular mechanisms controlling sugar translocation and the timing between early embryo development and subsequent seed filling in cucumber (Cucumis sativus L.) remain poorly understood. Here, we report that alkaline α-galactosidase 2 (CsAGA2) is expressed in the placental vascular bundles and funicular phloem, and its encoded protein is responsible for the hydrolysis of raffinose family oligosaccharides (RFOs). We demonstrate that FUSCA3 (CsFUS3) activates, while Auxin Response Factor 9 (CsARF9) represses, CsAGA2 expression. The CsFUS3–Sucrose Non-fermenting-1 (Snf1)-Related Protein Kinase 1α1 (CsSnRK1α1) interaction further enhances CsAGA2 expression, while CsARF9 recruits the TOPLESS (CsTPL) corepressor and further weakens CsAGA2 expression. Transgenic CsAGA2-RNAi (RNA interference), CsFUS3-RNAi, csfus3-crispr, and CsARF9-OE (overexpression) lines showed severe seed abortion rates, likely caused by reduced sugar supply during embryo development and seed filling. These results demonstrate the critical collaborative roles of these proteins in delivering sugars for seed development. Our findings reveal that CsAGA2 is an essential enzyme that is switched on by CsFUS3 to precisely regulate embryo development and provide the vast quantities of sugars needed for seed filling in cucumber. As the seeds mature, CsARF9 dampens CsAGA2 expression to gradually reduce the sugar supply to seeds. Therefore, our data suggest that the CsFUS3 and CsARF9 sequentially regulate embryo development and seed filling via CsAGA2-mediated RFO catabolism in cucumber. Our findings provide a new strategy for manipulating seed filling to increase yield in the breeding process of seed crops.

Excessive irrigation in protected vegetable production often results in soil nutrient loss and groundwater contamination. Cucumber (Cucumis sativus L.) is a widely cultivated and important vegetable in the world and a sensitive plant to irrigation water supply. In order to obtain higher water use efficiency (WUE) and to assess the leaching loss of mineral elements under the current strategies of irrigation and fertilization in the production of protected crops, we conducted experiments with three irrigation levels, namely, normal (NI), optimized (OI), and deficit irrigation (DI), on cucumber in a solar greenhouse. The results indicated that the contents of nitrate–nitrogen (NO₃⁻–N) in the top soil layer increased significantly under the reduced irrigation condition (OI and DI) after two cultivation seasons compared with normal irrigation (NI). However, there were no significant differences in the contents of available phosphorus (A–P) and available potassium (A–K) between the three treatments in each soil layer during a single irrigation cycle and for the whole growth cycle. In addition, compared to the NI condition, reducing the amount of irrigation (OI and DI) decreased the amount of leaching of the soil mineral elements by more than half without jeopardizing the fruit yield of cucumber, particularly for DI. Under the three irrigation treatments, the economic yield of cucumber varied from 64,513 to 72,604 kg·ha⁻¹ in the autumn–winter season and from 89,699 to 106,367 kg·ha⁻¹ in the winter–spring season, but the differences among the treatments were not significant. Moreover, the reduced irrigation treatments (OI and DI) substantially improved WUE by 43.9% and 135.3% in the autumn–winter season, and by 82.2% and 173.7%, respectively, in the winter–spring season, compared to the NI condition. Therefore, deficit or optimized irrigation was a potential and suitable irrigation strategy in the solar greenhouse for increasing the water use efficiency, reducing the amount of leached soil mineral elements, and maintaining the economic yield of cucumber crop. Overall, our results provided some insight into the future applications of water-saving irrigation techniques in sustainable greenhouse vegetable production.

Parthenocarpy is a pivotal trait that enhances the yield and quality of fruit crops by enabling the development of seedless fruits. Here we unveil a molecular framework for the regulation and domestication of parthenocarpy in cucumber (Cucumis sativus L.). We previously discovered a natural non-parthenocarpic mutant and demonstrated that the AP2-like transcription factor NON-PARTHENOCARPIC FRUIT 1 (NPF1) is a central regulator of parthenocarpy through activating YUC4 expression and promoting auxin biosynthesis in ovules. A Phe-to-Ser substitution at amino acid residue 7 results in a stable form of NPF1 that is localized in the nucleus. An A-to-G polymorphism (SNP-383) within an NPF1-binding site in the YUC4 promoter significantly enhances the activation of NPF1 towards YUC4, leading to an increased rate of parthenocarpy. Additionally, NPF1 influences bitterness by reducing cucurbitacin C biosynthesis through the suppression of Bt expression. Our results suggest a two-step evolutionary model for parthenocarpy and fruit bitterness during cucumber domestication.

Photosynthesis is critically dependent on both adequate light and nitrogen availability. In controlled agricultural environmentswith limited natural light, cucumber faces substantial challenges, including reduced photosynthetic efficiency and biomass accumulation.Nitrogen (N) is a vital component for the synthesis of various macromolecules, metabolites, and signaling molecules in plants. This studyassessed the impact of different nitrate levels (LL-N, LL-1/2N, and LL-2N) on the physiological, biochemical, and metabolic responsesof cucumber under low light conditions (80–100 μmol m–2 s–1 photosynthetic photon flux density) in comparison to standard light(400–500 μmol m–2 s–1 photosynthetic photon flux density) and unchanged nitrate. We observed that increasing nitrate levels to twicethe standard concentration (LL-2N) significantly enhanced shoot morphology, including weight and size, and improved photosyntheticperformance, stomatal conductance, and transpiration rates. In contrast, lower nitrate levels (LL-1/2N) exacerbated stress, leading todiminished photosynthesis and biomass. Biochemical analyses indicated that elevated nitrate concentrations activated key enzymesinvolved in the metabolism of carbon and nitrogen, including glutamine synthetase, sucrose phosphate synthase, nitrate reductase, andglutamate synthase. This led to improvement in the amounts of sucrose, fructose, soluble sugars, free amino acids, and soluble proteins.Additionally, the activities of antioxidant enzymes including superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase wereconsiderably greater under conditions of increased nitrate, which reduced the levels of malondialdehyde and H2O2 as oxidative stressmarkers. Furthermore, enhanced nitrate levels upregulated critical photosynthetic genes (rbcL, rbcS, FBPase, and SBPase mRNA),supporting the RuBisCO contents and overall photosynthetic capacity. These findings underscore the effectiveness of strategic nitrateenrichment in mitigating low light stress, providing essential insights for optimizing cucumber cultivation in controlled environmentsand improving productivity.

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.

Central metabolism produces carbohydrates and amino acids that are tightly correlated to plant growth and thereby crop productivity. Malate is reported to link mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. Although the function of malate metabolism-related enzymes in providing carbon has been characterized in some plants, evidence for this role in the fleshy fruit of cucumber is lacking. Here, radiolabeled bicarbonate fed into the xylem stream from the cucumber roots was incorporated into amino acids, soluble sugars, and organic acids in the exocarp and vasculature of fruits. The activities of decarboxylases, especially decarboxylation from NADP-dependent malic enzyme (NADP-ME), were higher in cucumber fruit than in the leaf lamina. Histochemical localization revealed that CsNADP-ME2 was mainly located in the exocarp and vascular bundle system of fruit. Radiotracer and gas-exchange analysis indicated that overexpression of CsNADP-ME2 could promote carbon flux into soluble sugars and starch in fruits. Further studies combined with metabolic profiling revealed that the downregulation of CsNADP-ME2 in RNA interference (RNAi) lines caused the accumulation of its substrate, malate, in the exocarp. In addition to inhibition of glycolysis-related gene expression and reduction of the activities of the corresponding enzymes, increased amino acid synthesis and decreased sugar abundance were also observed in these lines. The opposite effect was found in CsNADP-ME2-overexpressing lines, suggesting that there may be a continuous bottom-up feedback regulation of glycolysis in cucumber fruits. Overall, our studies indicate that CsNADP-ME2 may play potential roles in both central carbon reactions and amino acid metabolism in cucumber fruits.

Summary: Amino acid transporters are the principal mediators of organic nitrogen distribution within plants and are essential for plant growth and development. Despite this importance, relatively few amino acid transporter genes have been explored and elucidated in cucumber (Cucumis sativus). Here, a total of 86 amino acid transporter genes were identified in the cucumber genome. We further identified Amino Acid Permease (AAP) subfamily members that exhibited distinct expression patterns in different tissues. We found that the CsAAP2 as a candidate gene encoding a functional amino acid transporter is highly expressed in cucumber root vascular cells. CsAAP2 knockout lines exhibited arrested development of root meristem, which then caused the delayed initiation of lateral root and the inhibition of root elongation. What is more, the shoot growth of aap2 mutants was strongly retarded due to defects in cucumber root development. Moreover, aap2 mutants exhibited higher concentrations of amino acids and lignin in roots. We found that the mutant roots had a stronger ability to acidize medium. Furthermore, in the aap2 mutants, polar auxin transport was disrupted in the root tip, leading to high auxin levels in roots. Interestingly, slightly alkaline media rescued their severely reduced root growth by stimulating auxin pathway.