A novel cucumber albino mutant caused by chloroplast development deficiency

Background Photosynthesis is a fundamental process for plant growth and development dependent on a precise network, including formation of chloroplast and chlorophyll synthesis. Chloroplast development deficiency could lead to albinism in higher plant. Results Here, we report a cucumber albino recessive mutant that processed white cotyledons under light condition and is unable to produce first true leaf. Meanwhile, albino mutant could grow out creamy green cotyledons under dark condition but died after exposing to light. Using fluorescence microscopy and transmission electron microscope (TEM), impaired chloroplasts were observed. We identified 7 and 3 differentially expressed genes (DEG) involved in Chlorophyll metabolism and Methylerythritol 4-phosphate (MEP) pathway through transcriptome analysis, respectively. We also examined the reported homologous genes for albino mutants from other plants. Two of 12 genes, TOC159 and DXS1, were up-regulated in cucumber albino mutants as well. The reliability of RNA sequencing results were further confirmed by real-time quantitative PCR (qPCR). Conclusions Taken together, we elaborate the differences between albino mutant and normal seedlings from a single cucumber progeny. This mutant is a new material to study protoplast development. MEP, CDP-ME, ME-cPP, HMBPP, IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate.

chlorophyll content of cotyledon from wild type, albino mutants that grow under light and dark condition using fluorescence microscopy. Much intensive chlorophyll signals were observed in wild type compared with albino mutant that grown under dark conditions ( Fig. 2e; Presented in red). As expect, no chlorophyll signals were caught in albino mutants that grow under light condition.
Since most chlorophyll content in plants are presented in chloroplast, we further investigate the chloroplast ultrastructure in cotyledon of wild type and albino mutant that grew under both light and dark condition using transmission electron microscopy. In wild type cotyledon, we observed numerous well-developed, crescent-shaped chloroplast with stroma thylakoids, grana thylakoids, starch granules and plastoglobuli within the membranes (Fig. 2f1,4,7). In contrast, the chloroplast in albino mutant decreased dramatically in number and showed abnormal shape that lack of stroma thylakoids and grana thylakoids structure but contained osmiophilic plastoglobulis in inner membrane system (Fig. 2f2,5,8). The albino mutant that grow under dark condition comprised stroma thylakoid as well as osmiophilic plastoglobulis (Fig. 2f3,6,9).
To summarize, the above results indicated that the chloroplast development is impaired in the albino mutant under light condition during seedling development. Moreover, light probably acts as a lethal factor to the albino mutant by interrupting thylakoid biogenesis, as we could observe the presence of thylakoid in the albino mutant grown in dark but not under light condition.

Transcriptome profiling and identifying differentially expressed genes (DEGs) between albino mutant and wild type
The transcriptomes of cotyledons from albino mutant and wild type were examined using RNA-seq, each with three biological replicates. Overall, 97,869,948 to 123,516,412 clean reads were obtained after filtering low quality reads (Table 1). After mapping to the cucumber reference genome 9930 v2 (17,18), in total, 21,664 transcripts were detected. High coefficients among the replicates demonstrated the consistency of the transcriptional changes within each type (Fig. 3a). In total, 1,256 genes were found up-regulated and 1,584 were down-regulated in albino mutant compared with wild type (|log2FC|≥2) (Fig. 3b, Additional file 3: Table S3). Based on the annotation, DEGs were categorized to GO and KEGG pathway to deeply understand the significant biological processes and pathways between albino mutant and wild type. 2,175 DEGs were classified into 814 GO processes involved in three categories, biological process, cellular component and molecular function (Additional   file 4: Table S4), with biological process being dominant category. Cellular carbohydrate biosynthetic process (GO:0034637, p=0.00010) and Cellular carbohydrate metabolic process (GO:0044262, p=0.00017) are the most significantly enriched processes (Additional file 8: Figure S1). As shown in  Table S4). Totally, 957 DEGs were assigned to 110 KEGG pathways (Additional file 5: Table S5), among which top 20 enriched KEGG pathways were illustrated in Fig. 4. Carbon metabolism (KEGG: csv01200), phenylpropanoid biosynthesis (KEGG: csv00940) and starch and sucrose metabolism (KEGG: csv00500) pathways occupied large proportion, with 69, 50 and 44 DEGs included, respectively (Additional file 5: Table S5).
No variant was found among these genes between mutant and wild type. Nearly no gene, except two, TOC159 (Csa4G001670) and DXS1 (Csa3G114510) were up-regulated in the mutant (Additional file 7: Table S7).

Validation of DEG expression by RT-qPCR
Eighteen DEGs (gene names were listed in Additional file 1: Table S1) with absolute log2(FoldChange) > 3 were randomly selected for RT-qPCR verification. Genes encoding peroxidase, isocitrate lyase, Glutathione S-transferase, malate synthase and many others were included. The expression of each gene was presented by relative expression (-ΔΔCt) using normal green seedling as control. The correlation between relative expression value of albino mutant and RNA-seq result (log2FoldChange) was calculated in EXCEL using CORREL function. The correlation factor between RT-qPCR and RNA-seq data was 0.9899, indicating a strong correlation. Overall, RT-qPCR validation indicates the reliability of RNA-seq result.

Discussion
Albinism happens among different living things varying from human beings, animals as well as higher plants (33-35). In this study, some albino seedlings were observed in the progenies from a selfpollinated cucumber fruit during the breeding selection period of an inbred line named g32. This cucumber albino mutant presented white hypocotyl finally presented lethal performance without developing first true leaf under light condition. In most studies, happening of albino phenotype in plants were caused by absence of chlorophyll and abnormal chloroplast development (27, 36, 37).
Absent chlorophyll signal and defective chloroplast development in albino mutant were in consistent with most albino mutant in this respect. In normal seedlings, ultrastructure of chloroplast is well presented with compactly arranged chloroplasts while few or even no chloroplast structure was seen in albino seedlings. Also, an abnormal chloroplast ultrastructure in albino mutant were observed lacking starch granules and thylakoids, but with osmiophilic plastoglobuli (Fig. 4). Osmiophilic plastoglobuli generally appears as a result of the degradation of thylakoid membranes under stresses (38), therefore we propose that light might act as an abiotic stress cue for albino mutant, leading to the degradation of thylakoid membranes.
Interestingly, we found that this cucumber albino mutant is different to fln1, rpl21c, emb, pds3, toc159, dxs1 and purd albino mutants 15, 20-32 since it could recover white cotyledon to green under dark condition. Similar phenotype was reported in Arabidopsis albino pap7-1 mutant (39, 40). Pap7-1 mutant was with albino cotyledon and finally died grown under light condition but undistinguishable with wild type grown in darkness (40). From the TEM analysis, chloroplasts with thylakoids were observed. Since pap7-1 mutant could be arrested under very dim light (40), we tried to culture the mutant to dark to let it develop but finally failed. Still it needs to be proved whether our mutant could produce plant architecture or even flower as pap7-1 grown under sucrose supplemented medium and dim light.
Differences in transcriptional level between albino mutant and wild type were also determined. Key genes involved in chlorophyll metabolism and MEP pathway and thylakoid related genes were differentially accumulated. Genes in chlorophyll metabolism were mainly up-regulated in the albino mutant, except POR. This increased expression of most genes might be a feedback regulation. POR is a light dependent key enzyme required for chlorophyll biosynthesis by catalyzing protochlorophyllide to chlorophyllide (41, 42). It has been reported that POR is crucial for plant growth and development since nonfunctional plants displayed reduced chlorophyll content and severe photoautotrophic growth defects (43, 44). The expression level of POR is lower in albino mutant than in wild type, indicating that albino mutant might be incapable in chlorophyll biosynthesis. MEP pathway is essential for the biosynthesis of photosynthesis-related compounds, such as carotenoids, chlorophylls, gibberellins and abscisic acid, which is vital important for plant development and metabolism(45). The mutation of genes in the pathway impaired biosynthesis of these compounds, disrupted chloroplast development, Isocitrate lyase and malate synthase are two unique enzymes to glyoxylate cycle, which was considered essential for postgerminative growth and seedling establishment (51). Under normal condition, glyoxylate cycle enzyme activities decreased rapidly as seedlings become photosynthetic but maintained a continuously high level when grown in dark (52)(53)(54). In this study, the isocitrate lyase (Csa2G420990) and malate synthase (Csa1G050360) are abundant in mutant than in wild type (Fig. 6). Wild type started autotrophic growth at this timepoint already, with a high activity of peroxidase (Csa4G285740) and chlorophyll A-B binding protein (Csa3G664560), while albino mutant still needed to utilize the storage because it is still not able to photosynthesize resulted from a lack of functional chloroplast. Glutathione S-transferase have been reported mainly function in response to biotic and abiotic stresses, such as oxidative stress (55), high/low temperature stress (56, 57) and different pathogen invasion (58-60). As well, a high concentration of glutathione S-transferase resulted in decreased chlorophyll content (61). When albino mutant is exposed to light, a lethal factor rather than a gift, the mutant presented (Csa4G304240, Csa4G303170) (Fig. 6) more glutathione Stransferase than wild type, this might alsopromote chlorophyll degradation.
Since all the seeds were planted under the same culture condition, we could exclude the possibility of environmental but genetic determination of this albino phenotype. The genetic analysis revealed that this albino phenotype was recessively controlled by a single locus. To trace the mutation origin, the previous generation of g32 was also evaluated. Phenotypic evaluation presented no albino performance in all the tested seedlings (data not shown), indicating that the mutation most probably occurred in previous generation in heterozygosis and albinism emerged in this generation. Albinism is not a desired phenomenon in plant breeding since it could affect plant growth as well as production.
However, this mutant is of great importance for us to detect new gene that involve in protoplast development.

Conclusions
In this study, a novel naturally occurring albino mutant was firstly reported in cucumber. We showed that the albino mutant had abnormal chloroplast and did not produce any chlorophyll, therefore, the whole seedling presented white color. Interestingly, we found that this mutant is different from the ones in other plant species, since this cucumber albino mutant could recover white cotyledon to green under dark condition. In addition, with transcriptome data, we detected that differentially expressed genes involved in chlorophyll metabolism, Methylerythritol 4-phosphate (MEP) pathway, as well as thylakoid development. Taken together, this mutant can be a new material to study protoplast development.

Plant materials
Cucumber inbred line g32 was used in this study. The inbred line was provied by Vegetable Research Institute, Guangdong Academy of Agricultural Sciences. The institute is also in responsible for the plant materials used in this study. Yu'e Lin undertakes the formal identification of the plant material. This material has not been deposited in any publicly herbarium. Seeds from a self-pollinated g32 cucumber fruit were soaked into water for 4 hours and then kept in the incubator with moderate humidity at 28℃ for germination. Thereafter, germinated seeds were planted in plug tray in the greenhouse of Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China. Dark treatment was performed in a black plastic bag covered homemade growth chamber. Seven-day-old wild type and albino mutant seedlings were used for phenotypic evaluation, fluorescence microscopy, transmission electron microscopy analysis and high-throughput RNA sequencing.

Fluorescence microscopy
The abaxial epidermises of cotyledons were used for fluorescence observation. The autofluorescence (red) of chloroplasts were captured under Zeiss LSM710 confocal microscope at the following setting: excitation at 633 nm, emission at 647-721 nm. Data were analyzed using ZEN2010 software.

Transmission electron microscope(TEM)
Cotyledons of wild type and albino mutant seedlings grown under light condition, and cotyledons of albino mutant seedling grown in dark were analyzed for TEM. All the cotyledon samples were cut into 1-2 mm 2 sections and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in phosphate buffer (pH 6.8-7.2) under a vacuum for 3 hours, followed by washing with phosphate buffer. Subsequently, samples were fixed in 1% osmium tetroxide for 3 hours, followed by washing with phosphate buffer, and dehydrated in a graduated ethanol series. The samples were infiltrated with an increasing ratio of Spurr's resin dilutions (25%, 50%, 75%, and 100% (v/v)) to substitute ethanol. Finally, samples were embedded on Spurr's resin. After cutting, the sections were viewed under a HitachiH-7700 (Hitachi) transmission electron microscope.

RNA extraction
Total RNA was extracted from cotyledons of 7-day-old wild type and albino mutant seedlings using Trizol Kit (Promega, USA) according to the manufacturer's instructions. Extracted RNA was treated with RNase-free DNase I (TaKaRa, Japan) to remove residual DNA. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). cDNA library construction, high-throughput sequencing and mapping 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. Library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Thereafter, reads with adaptors, reads with unknown bases as well as low quality reads were removed to generate clean reads. The remaining high-quality clean reads were mapped to Cucumber (Chinese Long) Reference  Table S7. Differentially expressed genes of reported albino genes between albino mutant and wild type.
Additional file 8: Figure S1. Histogram of GO enrichment analysis. Growth phenotype of albino mutant and wild type from the progenies of a cucumber inbred line g32. a Differences in cotyledons of albino mutant and wild type seedlings. b Morphological difference between albino mutant and wild type seedlings.

Figure 2
Short-lived chloroplast recovery in albino mutant under dark condition. a1-a3 Schematic illustration of light and dark treatment of WT and albino seedlings. a1 Plants were treated with continuous dark before and after they emerged from substrate. a2 First, plants were treated with light, then after emerging from substrate, they were moved to dark. a3 First, plants were treated with dark, then after emerging from substrate, they were moved to light condition. b Phenotype of WT and albino seedlings under indicated condition that described in a1. c Phenotype of WT (c1) and albino (c2) seedlings in the indicated conditions that described in a2. d Phenotype of WT and albino seedlings in the indicated conditions that described in a3. d1 Phenotype of WT and albino seedlings exposed to light after 5 hours. d2 Phenotype of WT and albino seedlings exposed to light after 30 hours.