Photosynthetic contribution and characteristics of cucumber stem and petiole

doi:10.21203/rs.3.rs-640048/v1

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Photosynthetic contribution and characteristics of cucumber stem and petiole

https://doi.org/10.21203/rs.3.rs-640048/v1

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Background

Photosynthesis of plant non-leaf blade green tissue has been studied in some plants, but the photosynthesis characteristics of stem and petiole are poorly understood. Cucurbitaceous plants are climbing plants, and have a large biomass of stem and petiole. Understanding the photosynthetic contribution of cucumber stem and petiole to growth and the underlying molecular mechanisms are important for the regulation of growth in cucumber production.

Results

Here, the photosynthetic capacity of cucumber stem and petiole were proved by ¹⁴CO₂ uptake. The total carbon fixation of stem and petioles is around 4% to that of one leaf blade in cucumber seedling stage, while the proportion of carbon accumulated in stem and petioles redistributed to sink organs (root and growing point) is increased obviously under leaf less condition. Photosynthetic properties of cucumber stem and petiole were studied using a combination of electron microscopy, chlorophyll fluorescence imaging and isotope tracer to compare with leaf blade using two genotype of cucumber (dark green and light green stems). Compare with leaf blade, chlorophyll contents of cucumber stem and petiole are lower, and accompanying with lower chloroplast number, lower stoma number, but with higher thylakoid grana lamella number and larger stomata size. The total photosynthetic rate of stem and petiole is equivalent to 6 ~ 8% of one leaf blade, but the respiration rates were simiar in all the three tissues, which shown an almost 0 net photosynthetic rate in stems and petioles, and with lower non-photochemical quenching (NPQ). Transcriptome analysis showed that compared with leaf blade, there are significantly different gene expressions in photosynthesis, porphyrin and chlorophyll metabolism, photosynthetic antenna proteins and carbon fixation in stem and petiole. Although with lower Rubisco expression level in stem and petiole, Rubisco and PEPC enzyme activities were both higher in stem and petiole than in leaf blade, suggesting the photosynthetic and respiratory mechanisms in stem and petiole are different from those in leaf blade.

Conclusions

In this study, we confirmed the photosynthetic contribution to growth of cucumber stem and petiole, and shown their similar photosynthetic pattern in tissue anatomy, molecular biology and physiology.

Plant Molecular Biology and Genetics

Cucumber

Stem

Petiole

14C

Photosynthesis

Photosynthesis is the most basic physiological and biochemical metabolic pathway of plants, which is of great significance for plant growth and development. The main organ of photosynthesis in plants is leaf blade, but a large number of studies have shown that non-leafy green tissues such as petioles, stems and green fruits also have the basic anatomical structure and physiological characteristics of photosynthesis, and has a certain contribution to yield formation [1], such as celery petioles and tobacco stems [2], cucumber fruits and immature tomato fruits [3-5], cotton bolls [6], and even fruit subsidiary tissues, including wheat awn [7] and corn husk [8]. When corn bracts were removed 15 days after flowering, the yield decreased by 17.7% [9]. It is considered that sepal is the main photosynthetic organ of sunflower (Helleborus viridis L.), and the contribution rate of photosynthate to the whole plant is more than 60% [10]. Some of the non-leafy organs not only play an important role in plant growth and yield formation, but also in the abiotic stress conditions. In dry and hot ecosystems world-wide, many woody plants with photosynthetic stems are decline leaf area and increasing stem photosynthetic activity along aridity gradients [11, 12]. This suggests that photosynthetic stems convene some physiological advantages, such as extra carbon fixation capacity, improved water-use efficiency (WUE) during periods of many plants are leafless, and balance of respiratory costs due to reassimilation of CO₂ [13-19, 1, 20]. Cucumber is a climbing plants with a large biomass of stem and petiole. The old leaves in cucumber become sink organs and are susceptible to diseases and insect pests. Also, leaf blades are delicate compare to cucumber stems and petioles. Get rid of cucumber old leaves is one of the regular management to prevent cucumber plants from disease, and can improve the level of cucumber yield during middle and late harvest stage. Therefore, it is necessary to pay attention to the photosynthetic capacity of non-leaf green organs (stem and petiole) of cucumber both under normal and lack of leaf blade condition, which as an important supplement to the production capacity of leaf photosynthetic [1].

The main questions that we address in this paper are as follows. Are stem and petiole in cucumber have similar photosynthetic structure and function compare to leaf blade? What’s the photosynthetic contribution rate of cucumber stem and petiole compare to leaf blade, and how much of them contribute to sink organs? What’s the difference in photosynthetic gene expression between cucumber stem, petiole and leaf blade? We hypothesized that the photosynthesis is similar in stem and petiole but different from leaf blade, and the carbon distribution shifts when plants are leafless. In the future, we expect that cucumber stems and petioles can be adjusting to leaf blades in carbon acquisition and functional traits, but can maintain their physiological advantages such as stress resistance and so on.

¹⁴C feeding in cucumber stem and petiole

To proof the presence of photosynthesis in cucumber stems and petioles, and its allocation of assimilation, ¹⁴CO₂ were feeding under light to different organs of cucumber in seedling stage (Additional file 1: Fig. S1). The different tissues of cucumber irradiance were read at 48 h after a 1 h ¹⁴C labeling period. Although at a low level, a positive photosynthetic capacity of stem and petiole were shown, the assimilation of total ¹⁴CO₂ by stem and petioles is equal to 5.00% and 3.41% to one leaf blade in one seedling stage cucumber plant, and the total of non-leaf organs is 7.69%, which is nearly to the sum of petioles and stem (Table 1). Over half of the assimilated ¹⁴CO₂ (61.55% and 55.26%) were remained in fed stem and petioles after 48 hours, however the percentage of ¹⁴C remained in leaf blade is only 14.39%. These results shown that stems and petioles were both important source and sink organs under the absence of leaf blade condition, and they are the most important tissues to maintain growth after cutting off leaf blades.

The photosynthetic organs allocated ¹⁴C to growing point with a much lower proportion in leaf blade (0.08%) than in stem (2.06%) and petiole (2.36%), and all the growing point ¹⁴C feeding activity is around 1.5 kDPM, but the carbon distribution to roots were quite different with a high carbon distribution from leaf blades (26 kDPM) and low from stem (1.6 kDPM) and petiole (1.9 kDPM) under leaf less condition. These results may because the demand for photosynthetic products in growing point is saturated with the high level of carbon fixation in leaf blade, stem and petiole, but the ¹⁴C assimilation transport to root is insufficient for root growth after leaf cutting. The above results shown cucumber stem and petiole have the capacity of photosynthesis, and the photosynthetic product can contribute to sink organs growth with sufficient (growing point) or insufficient (root) supplement.

Ultrastructure content of cucumber stem and petiole

To explore the photosynthetic characteristics of cucumber stem and petiole, two genotypes, one dark green (DG) and one light green (LG) were used for further analysis (Fig. 1A). The chlorophyll and carotenoid content in DG is much higher than in LG (Fig. 1B) in stems and petioles (about 2-3 times) and also have a difference in leaf blade. The amount of chlorophyll and carotenoid in stems and petioles were much lower than leaf blades, and is about 4~8% of equal weight leaf blades.

In order to further explore the basic physiological structure of stems and petioles, we observed the Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) to observe the features of stomata and chloroplast in cucumber leaf blades, stems and petioles. Stomatal frequency on the surface of stems and petioles are only 3.12% and 1.32% of that on leaf blade, respectively (Additional file 2: Table S1), but the sizes of stomata are significantly larger on stem and petiole surfaces than those on leaf bade (Fig. 2). These percentages and the phenomenon of large stomata are similar to those in cucumber green fruit exocarp, and other species with photosynthetic in non-leaf organ [5, 21]. There is no significant difference between DG and LG on the stomata features and frequency (Additional file 2: Table S1).

Cells in stems and petioles are larger than in leaf blades, but the chloroplasts quantity per unit area in these tissues are lower (Fig. 3). The sizes of chloroplasts in stems and petioles are smaller (Fig. 3), and the number of thylakoid grana lamellae are 1.4 and 1.7-fold larger than in leaf blades (Additional file 2: Table S1), which is similar with cucumber fruit chloroplasts, and resembling the chloroplasts of shade plants [5]. Interestingly, there is no significant difference between DG and LG on the chloroplasts frequency, but the number of thylakoid grana lamellae is significant lower in different tissues of LG than in DG (Fig. 3, Additional file 2: Table S1).

Photosynthetic rate and chlorophyll fluorescence imaging

The photosynthetic rates of leaf blade, stem and petiole of DG and LG were measured by LI6400 portable photosynthetic measurement system. The net photosynthetic rate and respirator rate of leaf blade were almost same in both DG and LG cucumber, and calculated with a similar net photosynthetic rates around 18.7 μmol m^–2s^–1 (Table 2). Although the net photosynthetic rate in both stem and petiole were in a low level, and even got the negative data in stem, the total photosynthetic rate can be calculated by minus respiratory rate, and shown the positive photosynthetic capacity in stems and petioles. The net photosynthetic rates of stem and petiole in DG cucumber were significantly enhanced compared with that of LG, but there was no significantly difference in the respiration rate between theses varieties (Table 2).

Spatial heterogeneities of photochemical reactions in different tissues were identified using chlorophyll fluorescence imaging system [22]. PSII photochemical efficiencies (Fv/Fm) and Photochemical quenching coefficient (qP) of two varieties in different green tissues were almost similar (Fig. 4), indicating that the photosynthetic machinery for absorption and conversion of light energy are similar in different green tissues of cucumber. NPQ in cucumber stems and petioles were significantly lower than in leaf blades, and in petioles LG is slightly lower than DG, this may be related to their carotenoid contents in different plant tissues, which are responsible for heat dissipation.

Transcriptomic analysis of cucumber stem and petiole

In order to further focus on the photosynthetic and its product transport in the different green tissues of cucumber, RNA-Seq was used to analyze the transcriptomes of leaf blade, stem and petiole in DG cucumber. A total of 291.84 million clean reads for each sample was generated (Additional file 2: Table S2). After removing low-quality sequences, adaptor sequences and sequences contaminants, 561.87 million cleaned reads (96.26% of the sequenced reads) were mapped to the cucumber genome. For further comparative analysis, gene expression levels were calculated using the Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) reads approach. Principal component analysis (PCA) revealed that the stem, petiole and leaf blade could be classed into three separate groups (Fig. 5A) (Additional file 2: Table S2), indicating that there were great differences in gene expression among leaf blade, stem and petiole, and the samples have good repeatability.

A total of 15244 genes was expressed in the sampled tissues accounting for 65.57% (15244/23248) of the annotated genes (23248) in the cucumber genome [23]. About 52.77% (12267) of the annotated genes were present in all samples (FPKM≥1; Additional file 2: Table S3), and 8.38% (1277) were specifically expressed in stem and petiole. KEGG enrichment analysis shown that the specifically stem and petiole expression genes are associated with responses to alpha-Linolenic acid metabolism, plant hormone signal transduction, linoleic acid metabolism and so on.

KEGG enrichment analysis of the specific expression genes in stem (453), petiole (370) and leaf blade (465) shown that the specific expression genes in leaf blade are most related to plant hormone signal transduction (Fig. 5B and D); the specific expression genes in stem are most related to phenylpropanoid biosynthesis (Fig. 5B and E); the specific expression genes in petiole are most related to Nitrogen metabolism, photosynthesis, carotenoid biosynthesis and so on (Fig. 5B and F). We further searched the genes related to photosynthesis and carbon metabolism in these three groups of genes (Additional file 2: Table S4). In addition to common expression genes, no photosynthesis related gene specific expressed in stem and petiole, but there were some genes specific expressed in leaf blade, and most of them are related to PSII reaction center proteins, including PSII reaction center protein Z and H, PsbY (FPKM≥1) and so on. Some other genes involved carbon metabolism also specific expressed in leaf blade including 2 alpha carbonic anhydrase 7 genes. The pyruvate dehydrogenase E1 component subunit beta-3 gene specific expressed in stem, and an alpha carbonic anhydrase 7 gene specific expressed in petiole. These results shown the carbon metabolisms working in leaf blade, stem and petiole may different, and carbon metabolism is mainly carried out in the leave blade, the petiole and stem serve as supplements to leave blade.

The up-regulated and down-regulated genes compare with leaf were identified (Fig. 2B and C), and the common up regulated genes in stem and petiole were 1911 (1911/23248=8.22%), the common down regulated genes were 2314 (2314/23248=9.95%). KEGG analysis shown the common up-regulated genes are related to plant hormone signal transduction, alpha-Linolenic acid metabolism, biosynthesis of amino acids and so on in stem and petiole (Fig. 2D). These up-regulated genes are in the similar biological pathways with the specific genes expressed in stem and petiole, and shown the specific characteristics of stem and petiole compared with leaf blade (Fig. 2D, Additional file 2: Table S4). Most of the common down-regulated genes in stem and petiole are related to photosynthesis and the photosynthetic metabolic pathway, including porphyrin and chlorophyll metabolism, glyoxylate and dicarboxylate metabolism, carbon metabolism, photosynthesis-antenna proteins and so on, which are also in similar metabolism pathway with the specific genes expressed in leaf blade (Fig. 2E, Additional file 2: Table S4).

In addition, through the cluster heatmap of up-regulated and down-regulated genes in stem and petiole relative to leaf blade (Additional file 1: Fig. S2), we can see that the expression of differential genes (S1-S3) in stem and petiole (P1-P3) are obviously different from that of leaf blade, which indicates that the expression pattern of differential genes in stem and petiole are similar, but different from leaf blade. At the same time, it also shows that the three repeated samples we take have a good consistency.

Gene expression and enzymatic activity of Rubisco and PEPC

Rubisco enzyme and PEPC enzyme are key enzymes in photosynthesis, stomatal opening and initial immobilization of CO₂. Quantitative real-time PCR of Rubisco large subunit CsRbcL, small subunit CsRbcS, and PPC families including CsPPC1, CsPPC2 and CsPPC3 were measured in DG leaf blade, stem and petiole (Fig. 7). The expression of CsRbcL and CsRbcS in leaf was significantly higher than that in stem and petiole, while the expression of CsPPC1 and CsPPC3 in leaf blade was no-significant difference between different tissues. CsPPC2 expression level in leaf blade was significantly higher than that in stem and petiole, which is assistant with previously study that CsPPC2 expressed mainly in leaves [5].

The enzyme activities of Rubisco and PEPC in non-leaf organs (stem and petiole) were both higher in stem and petiole than in leaf blade (Fig. 7), which shown an opposite trend of Rubisco expression pattern. The content of Rubisco enzyme in leaves was very high, accounting for more than 50% of soluble protein [24], which may cause the decrease of enzymatic activity in units of total protein.

The contribution of cucumber stem and petiole to sink organ C accumulation

The non-leaf green organs of many plants have basic anatomical structures and physiological characteristics for photosynthesis, which can not only contain or produce chlorophyll, but also have actual or potential photosynthetic capacity, and has a certain contribution to the carbon fixation capacity and improve plant yield [25]. Cucumber petioles and stems are the main transport tissues for photosynthetic products in the growth of cucumber plants, but these parts themselves contain chlorophyll and can also undergo photosynthesis, which were confirmed by ¹⁴C tracer analyses in this study (Fig. 1), and are considered important strategies for additional C acquisition [25, 1].

Until now, we know little about how much of the non-leaf green organs contributed to carbon fixation and if they are important in maintain growth, especially under leafless condition in climbing plants like cucumber, which has a large proportion of stem and petiole biomass. ¹⁴C labeling results shown a positive carbon fixation capacity of stem and petiole and the total of non-leaf organs is equal to 7.64% to one leaf blade, which is sufficient for growing point but insufficient for roots carbon demand in seedling cucumbers under leafless condition. Also, the data shown a strong carbon distribution and consumption of stem and petiole in order to sustain growth in all of the treatments, indicating that cucumber stems and petioles are both sink organs and source organs (Table 1). The proportion of assimilated carbon allocated to growth point and roots are increased under leafless condition, indicating the changing of source-sink relationship in stems and petioles. These results indicated that cucumber stem and petioles not only can supplement to photosynthesis, but also play an important role in supporting growth under leafless condition.

The relationship between chlorophyll content and photosynthetic rate in cucumber stems and petioles

Although with a lower chlorophyll content of stems and petioles compared to leaf blades, the observation of stoma, chloroplast and grana lamella shown that they have the intact photosynthetic structure. ¹⁴C labeling experiment confirmed the photosynthesis ability of stems and petioles, but the photosynthetic structures between different tissues were significantly different, with a lower quantity of stoma and chloroplasts and a larger stoma size in stem and petiole than in leaf blade (Fig. 2). Moreover, those stem and petiole chloroplasts have relatively thicker grana lamellar of thylakoid membranes than leaf blade and the surface are covered with a thick layer of epicuticular wax (Fig. 2). This anatomical feature evidently to be able to reduce diseases and transpiration, but perhaps affects stomatal opening and closure, making them less sensitive to excess light and thermal dissipation, and results to a lower NPQ in stems and petiole (Fig. 4). Additionally, this may also relate to their lower carotenoids content, which are responsible for the protective mechanism of thermal dissipation capacity [26]. All these structure patterns (Fig. 3) are similar to cucumber fruits that have been discussed, which can minimize respiratory loss and allowing photosynthetic re-fixation of CO₂ before it can be released to the atmosphere [5].

The net photosynthetic rates of stem and petiole were higher in DG cucumber than LG, but the respiration rate were not (Table 2), which illustrate the net photosynthetic rate is related to chlorophyll content in stems and petioles, but respiratory rate is not. The petioles respiratory rates were lower than stems, resulted a positive net photosynthetic rate in petioles, and the negative data in stems may related to the thick diameter and cavity structure (Additional file 1: Fig. S1). The net photosynthetic rates and respiratory rates in leaf blades between the two varieties were not obvious, which may because too many chloroplasts in leaf blades and light saturation point were not reached.

Interestingly, the photosynthetic rate, chlorophyll content, chloroplast density and stomatal density of cucumber stem and petiole to leaf blade were all around 3%~9%, but the respiratory rate were 90% and 50% to leaf blade, respectively. An obviously phenomenon was shown that the low level of chlorophyll content, chloroplast and stomatal density in cucumber stems and petioles consistent with their lower photosynthetic rate. The net photosynthetic rate of stem was negative due to a higher respiration rate than photosynthetic rate, and the higher respiration rate in stem than petiole may because of its diameter is larger than petiole.

Cucumber stem and petiole have the similar Fv/Fm and qP value with leaf blade, suggesting their ability of light energy absorbed by the PSⅡ antenna pigment and photochemical electron transfer are at the same level, and the photosynthetic mechanism of stem and petiole for absorbing and converting light energy is well established.

The photosynthetic gene expression and enzymes activity of cucumber stems and petioles compare with leaf blades

Using physiological experiment data and RNA-seq data to analyze the photosynthesis of cucumber stem, petiole and leaf blade, it provides corresponding photosynthetic tissue anatomy, molecular biology and physiological evidence. Although in a lower expression level, stem and petiole have the basic photosynthetic genes expression including Rubisco, light-harvesting complexes (Lhca) of PSI and light-harvesting proteins (Lhcb) of PSII and so on. Some of the photosynthetic pathways genes expressed specifically in leaf blade such as PSII reaction center protein Z and H, and so on (Additional file 2: Table S4). This indicates that stem and petiole have the basic photosynthetic genes expression, but the specifically expressed genes in leaf blade may play important roles in the photosynthetic metabolism between different tissues, and may play important roles in enhance photosynthetic capacity in leaf blade. One of the photosynthetic genes was found only expressed in leaf blade is psbY, which is an important protein in Photosystem II for the redox control of cytochrome b559, and is related to susceptible to photoinhibition [27], which may related to the higher NPQ in leaf blade than stem and petiole, and is consistent with the Fluorescence imaging results (Fig. 4).

Interestingly, most of the photosynthetic genes have a similar expression pattern in stem and petiole compare with leaf blade, and relative to leaf blade down-regulated happened in stem and petiole involve in carbon metabolism and photosynthesis pathways such as carbon fixation in photosynthesis, porphyrin and chlorophyll metabolism and other pathways. Considering the similar photosynthetic structure, photosynthetic capacity, chlorophyll content and enzyme activity, the down-regulated genes in stem and petiole relative to leaf blade are in according with their chlorophyll content and photosynthetic ability.

Both Rubisco enzyme and PEPC enzyme play important roles in photosynthesis, and display different tissue-specific expression patterns and enzyme activity in cucumber. RbcL is usually encoded by a single chloroplast gene, RbcS is encoded by a nuclear family of several genes, and are considered functionally equivalent in a given species [28]. The higher expression level of both RbcL and RbcS in cucumber leaf blade than stem and petiole is consistent with their chlorophyll content. But the enzyme activity is in an opposite trend which shown an excess of Rubisco enzyme exit in leaf blade compare to stem and petiole.

In photosynthetic tissues, PEPC is a key enzyme in C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways, participates in the exchange of carbon dioxide and water between the atmosphere and leaves through its role in guard cells. The activity of PEPC and Rubisco enzyme in cucumber non-leaf organs (stem and petiole) was higher than that in leaf blade, and shown that CO₂ could be well captured. A similar phenomenon has also been noted in cucumber fruit and tomato fruits, that speculate the main function of PEPC is to capture respiratory CO₂ to synthesize 4-carbon organic acids, and these organic acids are then decarboxylated, and finally provide the turgor pressure necessary for cell expansion [29, 30]. Unlike cucumber fruit, cucumber stem and petiole have a large cavity in them (Supplement Fig. 1B). The anatomical evidence suggests that direct fixation of ambient atmospheric CO₂ by Rubisco is important in cucumber stem and petiole, and is considered as an important strategy for C acquisition [25, 1], and these may relate to their physiological structure, photosynthetic structure and chlorophyll content. In vegetative organs, the vascular bundles are always surrounded by Chl-rich cells that possess high activities of phosphoenolpyruvate carboxykinase (PPCK) [31, 2]. Until now, we do not know if the CO₂ from decarboxylation of organic acids is then re-fixation by Rubisco like the inside of cucumber fruit, or release into the air through the vascular tissue system or the cavity in cucumber stem and petiole.

Although leaf blade is the main organ of photosynthesis, it is not the only organ for photosynthesis. The photosynthesis of stem and petiole is a supplement to leaf blade. In this study, we focused on the photosynthetic contribution of stems and petioles at cucumber seedling stage, further study is needed at the cucumber climbing period, which has a large biomass of stem and petiole and may removing old and diseased leaf blade to adjust the source-sink relationship. Overall considering the contribution of cucumber stems and petioles to photosynthesis may play more important roles in the middle and late harvest stage of cucumber. From these data, we would like to speculate that leaving petioles when removing old and diseased leaf blades may help to supplement photosynthesis, which is the easiest way to increase individual photosynthesis and reduce disease of cucumber and other climbing plants.

This paper mainly provides relevant evidence of photosynthetic tissue anatomy, molecular biology and physiology for the photosynthesis of non-leaf organs in cucumber—stem and petiole. The contribution of non-leaf organ (stem and petiole) photosynthesis to plant carbon accumulation has been discussed and confirmed their photosynthetic complementary function under leafless condition. The study of cucumber non-leaf blade organs (stem and petiole) photosynthesis characteristics including photosynthetic structure, photosynthetic capacity, chlorophyll content, gene expression and enzyme activity, have been studied and shown a similar photosynthetic pattern in stem and petiole but different from leaf blade, which may provide a certain theoretical basis for improve cucumber photosynthesis.

Plant material and growth conditions

Two cucumber genotypes ‘Dark Green’ (‘DG’) and ‘Light Green’ (‘LG’) (cucumber seeds were acquired from Jiawang Li’s lab of Tianjin Kernel Cucumber Research Institute) were cultured in a phytotron with a 10 h photoperiod and a temperature cycle of 25/18℃ (day/night). The photon flux density (PFD) was 500 μmol m^–2s^–1. When plant had 5-6 true leaves, the third fully expanded leaf blades, counted from the shoot tip, the stems and the corresponding petioles were collected for analysis.

For ¹⁴C labeling experiment, ‘Dark Green’ seeds were germinated and the cucumber seedlings are growth in hydroponics with Hoagland nutrient solution. The plants were used for ¹⁴C labeling experiment when they had 5-6 true leaves.

¹⁴CO₂labelling

¹⁴CO₂ labelling were performed as described by Zhang et al. (2004) with modifications. Four treatments were conducted to detect carbon fixation capacity in a total of one plant for stem and petiole, one leaf blade, stem only and petioles only in the cucumber seedling stage. For stem and petiole samples, all the leaf blades were cut off and the stems and petioles were enclosed in plastic bags (Additional file 1: Fig. S1A). For leaf blade samples, the third cucumber leaf blades were enclosed in plastic bags, and all the other leaf blades were cut off (Additional file 1: Fig. S1B). For stem only and petioles only samples, all the leaf blades were cut off, and the aluminium-foil paper were used to cover the petioles or stems to expose corresponding stems (Additional file 1: Fig. S1C) or petioles (Additional file 1: Fig. S1D) part for photosynthetic under natural light condition. The radiolabel (1.85 MBq) was injected into a vial inside the bag, and ¹⁴CO₂ was released by the addition of excess 3 M lactic acid. After the leaf blades, stems and petioles were exposed to ¹⁴CO₂ for 1 h, an excess of 3 M KOH was injected into the vial to neutralize the acid and to absorb remaining ¹⁴CO₂. The bag was then removed, the translocation of radiolabel into roots and growing point were allowed to continue for 48h. Selected root, growing point, leaf blade, stem and petiole tissues were sampled and rapidly frozen in liquid nitrogen, the specific activity of ¹⁴C in each sample was analyzed by an LS 6500 Multi-Purpose scintillation counter. Three readings per treatments were replicated on three plants.

Determination of pigment contents

Leaf blade, stem and petiole samples form DG and LG cucumber varieties were taken and immediately frozen in liquid nitrogen. Pigment contents were measured according to standard methods [32, 33].

Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM)

Surfaces of leaf blade, stem and petiole were dissected for SEM observation, and sections of leaf blade, stem and petiole were sampled for TEM, from DG and LG cucumber varieties, respectively. Samples were fixed with 2.5% glutaraldehyde at 4°C for approximately 24 h, washed with PBS (pH 7.2) three times and post-fixed in 1% (v/v) OsO₄. The samples were then dehydrated through an ethanol series (30, 50, 70, 80, 90, and 100%).

For SEM, samples were critical-point dried using a desiccator (HCP-2; Hitachi), and coated with gold palladium (EIKO IB-3). Stomata were observed with a HITACHI S-3400 scanning electron microscope. Stomatal frequency was calculated in 5 different fields of view per sample.

For TEM, samples were embedded in Spurr’s resin. Ultrathin sections were cut with a LEICA UC6I microtome and viewed with a HITACHI-7500 transmission electron microscope [34]. The number of chloroplasts per unit area was calculated with 5 different microscope fields for each treatment.

Net photosynthetic rate (Pn)

Net photosynthetic rate (Pn) was measured using a LI-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA) equipped with an infrared gas analyzer (IRGA, 6400-02B). Cucumber plants were first adapted to gas exchange light intensity conditions (usually 1 h prior to the measurement), and then leaf blade, stem or petiole samples were prepared and immediately put into the leaf chamber. The net photosynthetic rate of sampled tissues was defined as the difference between CO₂ evolution rates under light and dark condition. Three readings per samples were replicated on three plants.

Chlorophyll fluorescence imaging

Chl fluorescence images were taken using an imaging fluorometer (FluorCam 1000-H, P.S.Instruments, Brno, Czech Republic). Plant material was first dark-adapted for 30 min at room temperature. A laboratory-built pneumatic shutter system provided 800 ms saturating light pulses (2000 μmol m^–2 s^–1 PFD) during a 1-min illumination with actinic light (500 μmol m^–2 s^–1 PFD). The opening and closing of the shutters were controlled by a laboratory-built digital interface connected to a personal computer. Color video images of PSII photochemical efficiencies (Fv/Fm) and Photochemical quenching coefficient (qP), and steady-state non-photochemical quenching (NPQ) were generated as described previously [35, 36].

Enzyme assay and immunoblotting

Enzymatic activities of the Rubisco and PEPC enzymes were measured as described previously [37, 38]. The activity of both enzymes was calculated by monitoring the decrease of NADH absorbance at 340 nm with a spectrophotometer (Unico UV-2802PC, USA).

RNA sequencing

Leaf blade, stem and petiole samples from DG plants were used for RNA sequencing, each for three biological replicates. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Raw data (raw reads) of FASTQ format were firstly processed through in-house perl scripts. The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then aligned to the cucumber genome [39] using Hisat2 tools soft. FPKM (fragments per kilobase of exon per million fragments mapped) values of the expression genes in different tissues were investigated and FPKM values less than 1 were treated as not accumulated and discarded. For each biological replicate, the detection procedure was executed completely and only the genes appearing in all three biological replicates were treated as final transmissible genes. Differentially expressed genes were identified with the DESeq package [40]. For further comparative analysis, GO enrichment and KEGG pathway analysis was performed using the OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools).

CsRbcL: cucumber rubisco large subunit; CsRbcS: rubisco small subunit; DEGs: differentially expressed genes; FPKM: fragments per kilobase of exon per million fragments mapped; Fv/Fm: maximum potential quantum efficiency of photosystem II; FW: fresh weight; KEGG: kyoto encyclopedia of genes and genomes; NPQ: non-photochemical quenching; PEPC: Phosphoenolpyruvate carboxylase; PPC: Phosphoenolpyruvate carboxylase gene; PPFD: photosynthetic photon flux density; PSII: photosystem II; qP: Photochemical quenching coefficient; Rubisco: Ribulose-1,5-bisphosphate carboxylase/oxygenase.

Acknowledgments

We thank Professor Zhenxian Zhang (China Agricultural University of Vegetable Science) for providing the initial idea of this paper, and thank for the support from collaborative innovation center of protected vegetables surround Bohai gulf region.

Authors' contributions

XL, XS and ZZ conceived and designed the experiments; WS, NM, and HH and performed the experiments; XL, and WS wrote the manuscript; JW, SM, HL, and SZ assisted in performing the experiments. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant nos. 31801850 to X.L.) and the The National Key Research and Development Program of China (2019YFD1000301-8 to X.L). The funding organizations played no role in this study.

Availability of data and material

The RNA-seq data has been submitted to NCBI under the accession number of PRJNA752681 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA752681).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of vegetable science, College of Horticulture, China Agricultural University, Yuanmingyuan Xilu 2#, HaiDian District, Beijing 100193, China.

²Tianjin Kerenl Cucumber Research Institute, 301 Baidi Road, Nankai District, Tianjin 300192, China.

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Table 1 Distribution of ¹⁴C photosynthate translocated in cucumber different tissues.

Treatment	Growing point	Petiole	Stem	Root	Leaf blade	Ratio to one leaf blade
Stem & Petiole (A)	0.94%±0.02%	33.38%±3.13%	59.78%±4.05%	5.90%±0.94%		7.64%
Leaf blade (B)	0.08%±0.02%	16.38%±1.29%	67.60%±5.79%	1.54%±0.13%	14.39%±1.23%	100.00%
Stem (C)	2.06%±0.13%	34.42%±8.26%	61.55%±7.09%	1.97%±1.03%		4.97%
Petiole (D)	2.36%±0.14%	55.26%±1.70%	39.08%±2.92%	3.29%±0.08%		3.39%

Table 2 Photosynthetic rate and respiration rate of cucumber Dark Green (DG) and Light Green (LG) cucumber.

Plant tissue	Net photosynthetic rate (μmol^-2 s^-1）	Respiratory rate (μmol^-2 s^-1）	Total photosynthetic rate (μmol^-2 s^-1）
DG Leaf blade	18.7±0.26a	-2.1±0.29b	20.8
LG Leaf blade	18.6±0.12a	-2.3±0.03b	20.9
DG Stem	-0.21±0.027c	-1.95±0.14b	1.74
LG Stem	-0.40±0.037d	-2.12±0.08b	1.72
DG Petiole	0.37±0.07b	-1.08±0.13a	1.45
LG Petiole	0.21±0.028bc	-1.37±0.13a	1.58

No competing interests reported.

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Photosynthetic contribution and characteristics of cucumber stem and petiole

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