Phenotypic characterization of a cucumber albino mutant
We observed a few natural occurring albino seedlings during the seed increase of cucumber inbred line “g32” which is a south china type cucumber. The cotyledons of these albino seedlings are small and absolute white, and of which hypocotyl is short and white and primary root length is short (Fig.1). The albino mutants dried out and died in a few days after emerging from substrate without growing any true leaves.
Inheritance model of albino mutant
To investigate the inheritance nature of the albino mutant, we planted a total of 123 seeds from the self-pollinated g23 cucumber, of which 116 were germinated (germination rate 94.3%). Among the germinated seeds, 32 and 84 seedlings present albino and wild type phenotypes which fit the Mendelian ratio for single recessive gene (χ2 = 0.414, p = 0.520, Additional file 2: Table S2).
Short-lived chloroplast recovery in albino mutant under dark condition
Occasionally, we observed some subtle greenish cotyledons in few albino mutants that were grown under the canopy. It draws our attention that albino mutation might be affected by light. We therefore performed three sets of experiments to validate this hypothesis (Fig. 2a). In the first experiment, all the seeds were grown under dark condition all the time. After emerging from substrate, albino mutant presented creamy green cotyledon and completely white hypocotyl and wild type seedlings show yellowish green cotyledon and hypocotyl (Fig. 2b). As continuedly dark environment, both albino mutant and wild type spindled and died without growing first true leaf eventually. In the second experiment, after the seeds emerged from substrate, we first let them grow under light condition until we are able to distinguish albino mutant and wild type. Later on, we transferred them under dark condition and found that albino mutant maintained whitish tissues without turning to cream green (Fig. 2c1-2). In the last experiment, the seeds grew under dark condition until seedlings emerging from substrate, and then they were exposed to light (Fig. 2a3). Upwards 5 hours after exposed to light, the green color in the cotyledon of albino mutant started to degrade (Fig. 2d1). The cotyledon contracted and dry out after exposed to light for 30 hours (Fig. 2d2). Therefore, we may conclude that light is a lethal factor for albino mutant and this damage is irreversible.
Most of the albino phenotypes in other plants are lack of chlorophyll. Thus, we examined the 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 Fig. 4, transmembrane transport (GO:0055085) and transporter activity (GO:0005215) accounted for the majority among the enriched GO terms, including 171 and 196 DEGs, respectively (Additional file 4: 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).
Differentially expressed genes involved in chlorophyll metabolism
Porphyrin and chlorophyll metabolism (KEGG: csv00860) was a significantly enriched pathway in KEGG analysis. Many key enzymes involved in this pathway showed distinct expression profile between albino mutant and wild type. Most of genes, including HEMB (Csa2G401270), HEME (Csa4G082410, Csa5G218840), HEMF (Csa4G056670), HEMG (Csa6G007980), CHLG (Csa4G311220) and CAO (Csa6G385090) were slightly higher expressed in albino mutant than in wild type (Fig. 5a, Additional file 6: Table S6). However, POR (Csa4G638340) was down-regulated in albino mutant, presenting opposite pattern to other genes (Fig. 5a, Additional file 6: Table S6).
Differentially expressed genes involved in Methylerythritol 4-phosphate (MEP) pathway
The methylerythritol 4-phosphate (MEP) pathway is a source for the production of isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in photosynthetic eukaryotes(19). As shown in Fig. 5b, there are at least seven key enzymes involved in the MEP pathway, four candidate genes encoded CMS (Csa3G113320, Csa4G049620), CMK (Csa1G600780) and MCS (Csa4G049620) were found down-regulated in albino mutant (Additional file 6: Table S6).
Thylakoid related genes are affected in albino mutant
Nineteen DEGs belonging to thylakoid related functional activities, i.e., thylakoid (GO:0009579), thylakoid part (GO:0044436) and thylakoid membrane (GO:0042651) were highlighted (Fig. 6, Additional file 3: Table S3). Three PsbPs (Csa2G030040, Csa1G088470 and Csa1G181310) were found up-regulated in albino mutant while other 16 genes PsbR (Csa4G064020), PsaG/PsaK (Csa3G060980, Csa6G525340), PsbO (Csa6G488340), PsaD (Csa3G147780), PsaH (Csa3G483830), PsbP (Csa4G063440), PsaE (Csa2G079660), PsbY (Csa5G592810), PsbW (Csa7G378440), PsaN (Csa6G483300), PsbQ (Csa1G066480, Csa3G414060), PsbX (Csa1G595840), PsaF (Csa1G714680) and PetM (Csa7G075020) were down-regulated in albino mutant (Additional file 3: Table S3).
Homologous genes from albino mutants in other species
Mutation of FLN1 (fructokinase-like 1) from barley and rice(20-22), RPL21c (chloroplast 50S ribosome protein L21) from arabidopsis and rice(23, 24), EMB (embryo-specific) from arabidopsis(25-28), PDS3 (phytoene desaturase) from arabidopsis(29), TOC159 (Translocase of chloroplast 159) from arabidopsis(30, 31), DXS1 (1-deoxy-D-xylulose-5-phosphate synthase 1) from tomato(15), and PurD (phosphoribosylamine-glycine ligase) from rice(32) could cause albinism in higher plant. The CDS of homologous genes of albino mutant and wild type were compared based on our transcriptome results. 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.