Hemerocallis Middebdorffii Trautv. & C. A. Mey and Hemerocallis ‘black-eyed stella’ and ‘stella de oro’ were cultured in experimental fields (Shanghai Institute of Technology, N: 30${}^{\circ }$50${}^{\prime }$33.98${}^{\prime }$${}^{\prime }$ E: 121${}^{\circ }$30${}^{\prime }$38.34${}^{\prime }$${}^{\prime }$), and each plant was grown in a single pot. The cross and self-pollinated progeny were cultured in chambers at 25 °C and 16 h light and 8 h dark photoperiod with cool white illumination (46.8 µmol photons$\cdot$m${}^{-2}$$\cdot$s${}^{-1}$). Plants were grown in soil and cultured in a medium containing half-strength Murashige and Skoog (MS) salts [41] and 3% sucrose (v/v), MS medium, sucrose, and other drugs were purchased at Shanghai Titan Technology Co., Ltd. (Shanghai, China).

The sections of daylily leaf tissue from albino mutants and wild-type plants were observed and recorded under a light microscope (ECLIPSE E200, NIKON, Japan).

Transmission electron microscopy analysis was performed according to the methods described by Hashimoto et al. [42] with some modifications. In detail, the leaf tissues of mutant and wild-type plant leaves were sampled, fixed and pumped with 4% glutaraldehyde at 4 °C, stored at 4 °C overnight and washed three times with 0.1 mol$\cdot$L${}^{-1}$ phosphate buffer (pH = 7). Then, the leaf samples were fixed with 1% osmic acid for 2 h and washed three times with phosphate buffer. The leaf samples were then dehydrated in a series of ethyl alcohol solution gradually followed by acetone, embedded in resin, and sectioned by slicing in an ultrathin slicing machine (EM UC7, LEICA, German). The samples were stained and then observed by transmission electron microscope.

The contents of chlorophyll and carotenoids were determined according to the methods described by Chen [43] with some modifications. The leaf samples of 20d daylily seedlings were ground using a pestle and mortar. Chlorophyll and carotenoids were extracted using a prepared extraction solution containing ethanol, acetone, and water in a ratio of 5:4:1 (v/v/v). The absorbance was measured by a microplate reader (Spark readers, TECAN, Swiss) at wavelengths of 470, 645 and 663 nm. The content of total chlorophyll, chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoids was calculated according to the following formula:

$$\begin{array}{c}\hfill \text{Chl (total Chlorophyll})=6.63\times \text{D\_663}+18.08\times \text{D\_645 (1)}\\ \hfill \text{Chla}=13.95\times \text{D\_663}-6.88\times \text{D\_645 (2)}\\ \hfill \text{Chlb}=24.96\times \text{D\_645}-7.32\times \text{D\_663 (3)}\\ \hfill \text{Car}=(1000\times \text{D\_470}-2.05\times \text{Chla}-114.8\times \text{Chlb})/245(4)\end{array}$$

The content of 5-aminolevulinic acid (ALA) was determined according to the methods described by Dei [44] with some modifications [45]. Leaf samples of 12-day-old daylily seedlings were ground and ALA was extracted by 4% trichloroacetic acid (m/v). Approximately 5 mL of extraction solution, 2.35 mL of sodium acetate (1 mol$\cdot$L${}^{-1}$), 0.15 mL of acetylacetone, and 2.5 mL acetate buffer (1 mol$\cdot$L${}^{-1}$, pH 4.6) were mixed and placed in boiling water for 10 min. The coloration of the mixture was performed by adding an equal volume of Ehrlich-Hg reagent after cooling. The absorbance was then measured by a microplate reader at 553 nm. The ALA content was then calculated according to the formula 7.2 $\times$ 10${}^4$$\times$ D${}_{553}$.

The content of porphobilinogen (PBG) was determined according to the methods described by Peng et al. [46] with some modifications. Leaf samples from 12 d old daylily seedlings were ground, and PBG was extracted by buffer containing 0.1 mol$\cdot$L${}^{-1}$ EDTA and 0.6 mol$\cdot$L${}^{-1}$ Tris-HCL (pH 8.2). Coloration of the extraction was performed by adding an equal volume of Ehrlich-Hg reagent. Then the absorbance was measured by a microplate reader at 553 nm. The content of PBG was calculated according to the formula: 6.1 $\times$ 10${}^4$$\times$ D${}_{553}$.

The contents of uroporphyrinogen III (urogen Ⅲ) and coproporphyrinogen III (coprogen Ⅲ) were determined according to the methods described by Bogorad et al. [47] with some modifications. Leaf samples of 12 d old daylily seedlings were ground, and PBG was extracted by 0.067 mol$\cdot$L${}^{-1}$ phosphoric acid buffer (pH 6.8). Then, 5 mL of extraction solution and 0.25 mL sodium thiosulfate were mixed and illuminated with intense light for 20 min. The pH of the mixture was adjusted to 3.5 by adding 1 mol$\cdot$L${}^{-1}$ glacial acetic acid. The absorbance of the aqueous phase, which was combined after chloroform extraction, was measured by a microplate reader at 405.5 nm. The urogen Ⅲ content was calculated using the formula: 5.48 $\times$ 10${}^5$$\times$ D${}_{405.5}$. The absorbance of the aqueous phase, which was combined after ether extraction, was measured by microplate reader at 399.5 nm. The content of coprogen Ⅲ was calculated according to the formula: 4.89 $\times$ 10${}^5$$\times$ D${}_{399}$.

The content of protoporphyrin IX (proto Ⅸ), Mg-protoporphyrin IX (Mg-proto Ⅸ), and pchlide were determined according to the methods described by Hodgins et al. [48] with some modifications [46]. Leaf samples from 12 d old daylily seedlings were ground after the addition of 25 mL of 80% alkaline acetone. The extraction was centrifuged at 15000 $\times$g and 4 °C for 15 min. The absorbance of the supernatant was measured by a microplate reader at wavelengths of 575, 590, and 628 nm. The content of proto Ⅸ, Mg-proto Ⅸ, and pchlide were calculated according to the following formula:

$$\begin{array}{c}\hfill \text{proto IX}=0.18016\times \text{A\_575}-0.04036\times \text{A\_628}-0.04515\times \text{A\_590 (5)}\\ \hfill \text{Mg – Proto IX}=0.06077\times \text{A\_590}-0.01937\times \text{A\_575}-0.003423\times \text{A\_628 (6)}\\ \hfill \text{Pchlide}=0.03563\times \text{A\_628}+0.007225\times \text{A\_590}-0.02955\times \text{A\_575 (7)}\end{array}$$

Photosynthetic parameters were determined according to the methods described by Iqbal et al. [45] with some modifications [49]. A portable photosynthesis tester (CIRAS-3, PP SYSTEMS, USA) was used to determine photosynthetic parameters. The fifth leaves from apical meristem of daylily were selected for measurement in September and October 2022 (10:00–12:00 am). Photosynthetic parameters including net photosynthetic rate (Pn), water use efficiency (WUE), transpiration rate (Tr) and intercellular CO${}_2$ concentration (Ci) were recorded after that the absolute value of the change amplitude of Pn was less than 0.2.

The relative water content of the leaves was determined according to the methods described by Chen et al. [50] with some modifications. Daylily leaves with the same physiological status were treated under drought conditions, and weighed. Fresh weight was recorded as the initial weight. The saturated weights (Wt) were recorded after the samples being placed in distilled water for 2 hours to absorb water. After that, the dry weights (Wd) were recorded after the samples being dried at 80 °C for 48 h. The water content (RWC) was calculated according to the following formula:

$$\mathrm{RWC}=(\mathrm{Wf}-\mathrm{Wd})/(\mathrm{Wt}-\mathrm{Wd})(8)$$

Soil moisture content was determined according to the methods described by Wang et al. [51] with some modifications. The wet weight (M1) of the soil was acquired by randomly weighing the soil from three different positions in the pot. Dry weight (M2) was recorded after the samples being dried at 80 °C for 48 h. The soil moisture (W) content was calculated according to the following formula: (M1 – M2)/M2 $\times$ 100%.

Leaf samples from 12 d old albino mutant and wild-type plants were ground, and five sets of biological replicates of each sample were randomly sampled. Three sets with the good-quality samples were obtained for analysis. Total RNA was extracted after digestion by DNase. The mRNA was enriched by magnetic beads with oligo dT. Used mirVana™ miRNA ISOlation Kit, Ambion-1561 kit and Refrigerated centrifuge (ST16R, Thermo, USA), Gel imaging system (Tanon 2500, Biotanon Co., Ltd., Shanghai, China), UV spectrophotometer (NanoDrop 2000, Thermo, USA).

cDNA libraries were constructed using a VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina sequencing according to the manufacturer’s instructions. The libraries were finally sequenced using an Illumina Novaseq 6000 platform. A total of 150 bp paired-end reads were generated. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Oligonucleotide adapters were removed using Trimmomatic software (version 0.36) [52], and clean reads were obtained after low quality bases and N bases were filtering out. Clean reads were then spliced through the paired-end method performed using Trinity software (version 2.4) to obtain transcript sequences [53]. The most extended sequence was selected as a unigene. The unigenes were annotated to the NR, COG/KOG, and Swissprot databases. GO annotation was acquired by mapping Swissprot ID to the GO term, and pathway information was acquired by comparing unigenes with the KEGG database [54].

Fragments per kilobase of exon model per million mapped fragments (FPKM) and unigene count were analysed in bowtie2 (version 2.3.3.1) [55] and eXpress software (version 1.5.1) [56]. The number of unigene reads in the samples were acquired by eXpress software. All the data were standardised by estimating SizeFactors, a function of DESeq package (version 1.18.0) [57]. The p-value and foldchange of the difference in gene expression between mutant and wild-type plants were calculated using nbinomTest software. The unigenes with difference value greater than 2 and p value less than 0.05 were selected for further analysis.

RNA-seq analysis was performed using Bioedit, NCBI, and Tair (https://www.arabidopsis.org/).

Six differentially expressed genes (DEGs) from RNA-seq data related to terpene, carotenoids, and chlorophyll metabolism were randomly selected for quantitative real-time PCR (qPCR) validation. Primers were designed using premier5 software (Supplementary Table 1). The Prime ScriptTM RT reagent kit with gDNA Eraser (TaKaRa) was applied to synthesise cDNA. qPCR was then performed according to the methods described by Zuo [58]. HfUBQ and HfEF-1a were used as internal reference genes [59]. The 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method was used for data analysis [50].

Two-week-old sterile cultured self-pollinated progeny of daylily ‘Black-eyed stella’ were transplanted to a medium containing half-strength MS salts, 3% sucrose (v/v), and 10% PEG2000 (v/v) [60]. One-year-old daylily plants, which were cultured in pot with soil, were left unwatered for two weeks, whereas the control plants were regularly watered every two days. Relative soil water content was determined at the same time.

At least three independent biological replicates were performed for each experiment. Statistical analysis was performed with Excel 2010 and SPSS26. Figures were made using Origin8 software.

Albino seedlings were observed during the selection and development of new varieties from self-pollinated progeny of daylily ‘Black-eyed stella’ and one of its parents, ‘Stella de oro’ in May 2020. The leaf colour of the albino seedlings was light yellow (Fig. 1A). The albino seedlings displayed a dwarf phenotype (Fig. 1B), and survived for less than 15 days in soil. However, these seedlings survived longer when grown on the medium containing half MS salts and 3% sucrose under sterile culture conditions (Fig. 1C–F). The chlorophyll and carotenoid content of albino seedlings was determined and compared with those of wild-type plants. The results showed that the content of chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoids decreased significantly compared with that of the wild-type plants (Supplementary Fig. 1). Albino phenotypes are generally attributed to changes in the metabolism of photosynthetic pigments and chloroplast development. Therefore, a microscopic analysis was performed to verify whether the chloroplast development of the mutant plants was altered. Extremely few chloroplasts were found in the albino mutant leaf cells (Fig. 1G,H). To better understand chloroplast development, transmission electron microscopy was used for investigating the morphology of mesophyll plastids. Both the number of thylakoids and the extent of stacking in the grana were significantly reduced in albino mutant plants (Fig. 2).

Fig. 1.

Comparison of the morphological and characterization of the plastid between albino and wild-type seedlings of daylily. (A) Seedlings of 15 d old wild-type (right) and albino mutant (left) seedlings cultured in the soil. (B) Height of seedlings of 15 d wild-type (left) and albino mutant (right) seedlings cultured in the soil. (C–E) Seedlings of albino mutant culture on the media containing half MS salts and sucrose under sterile conditions. (G,H) Light microscopy of the chloroplast of the leaf cells of albino mutant (G) and wild-type seedlings (H). The bars in (A–F) represent 1 centimeter, and in (G,H) represent 20 micrometers. Mean values $\pm$ SE (n = 3); significant values **p 0.01.

Fig. 2.

Microscopic analysis of the plastids tissue between albino mutant and wild type seedlings of daylily. (A,C,E) Transmission electron microscopic examination of plastids of albino seedlings. (B,D,F) Transmission electron microscopic examination of plastids of wild-type. The bars in (A,B) represent 10 micrometers, (C,D) represent 2 micrometers, and (E,F) represent 1 micrometer.

To understand the mechanism of the decrease in chlorophyll content, the precursors of chlorophyll and carotenoids biosynthetic pathways were measured. Except PBG, the content of the precursors of chlorophyll biosynthesis significantly decreased in the leaves of albino mutants compared with that of wild-type plants (Supplementary Fig. 3).

Daylily ‘Black-eyed stella’ is a cross between two diploid parents ‘Stella de oro’ and ‘Little celena’ (https://www.daylilies.org/DaylilyDB/). Albino mutants were isolated from the self-pollinated offspring population of ‘Black eyed stella’ and one of its parents, ‘Stella de oro’. The F2 population of ‘Black-eyed stella’ was used for further analysis, and the segregation ratio was in accordance with the expected Mendelian ratio of 3:1 (Table 1). Furthermore, all cross-pollinated offspring between H. ‘black-eyed stella’ and H. Middebdorffii Trautv. & C. A. Mey displayed a wild-type phenotype. However, albino seedlings accounted for approximately a quarter of the F2 population.

The surviving F2 plants should have consisted of two-thirds of heterozygous plants and one-third of homozygous plants, because the albino mutant plants died before flowering. The plants with self-pollination progeny that did not appear as albino seedlings were designated as homozygous plants (Alb${}^{+/+}$), and the other F2 plants were then marked as heterozygous (Alb${}^{+/-}$).

RNA-seq was performed to compare gene expression patterns between the mutant and wild-type plants. Then, 41.96 G clean data were acquired. The effective data volume of each sample ranged from 6.74 G–7.17 G. The Q30 bases were distributed in 95.47–95.55%, and the average GC content was 46.59%. A total of 53479 unigenes were spliced and had a total length of 55899490 base pairs (bp) and an average length of 1045.26 bp (Supplementary Table 3). These results showed that the quality of the RNA-seq data was good enough for subsequent analysis. A total of 7952 DEGs, including 4069 up-regulated genes and 3883 down-regulated genes were identified between daylily albino mutant and wild-type plants.

GO function enrichment analysis showed that many DEGs were involved in biological processes related to cellular components and molecular function, such as cellular process, metabolic process, cell part, organelle, membrane, membrane part, membrane-enclosed lumen, binding, catalytic activity, and transporter activity (Supplementary Fig. 2). KEGG significant enrichment analysis revealed that the DEGs were involved in 125 metabolic pathways. The metabolic pathways with the most DEGs were carbohydrate, lipid, amino acid, and energy metabolism. The down-regulated DEGs were pyruvate, amino sugar, nucleotide, starch and sucrose metabolism (Supplementary Fig. 3, Supplementary Table 4).

Six DEGs were randomly selected from genes encoding for terpene, carotenoid and chlorophyll metabolism and one transcription factor for quantitative real-time PCR (qPCR) validation, because all these genes were related to the albino trait (Fig. 3). The expression level of DEGs between qPCR and RNA-seq data was coincident (Supplementary Table 5). Therefore, the RNA-seq data described here can be considered reliable.

Fig. 3.

qPCR validation of DEGs obtained from RNA-seq data. DXS, PORA, DXR, CRTRSO, WRKY24 and CLH2 stand for daylily 1-deoxy-D-xylulose-5-phosphate synthase, protochlorophyllide oxidoreductase A, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, carotenoid isomerase, WRKY transcription factor 24 and chlorophyllase 2, respectively. Mean values $\pm$ SE (n = 3); significant values *p 0.05, **p 0.01; ns, no significance; qPCR, realtime quantitative polymerase chain reaction; RNA-seq, RNA sequencing.

Given that the level of chlorophyll and carotenoid in albino mutants were much lower than those of wild-type plants (Supplementary Table 2), the DEGs related to chlorophyll biosynthesis were analysed with the RNA-seq data, showing that most of the DEGs related to chlorophyll biosynthesis were down regulated (Fig. 4, Ref. [61, 62, 63]). The fold change range was 0.20–0.49 (Supplementary Table 6). CAO, a gene necessary for transforming Chla to Chlb in chlorophyll oxygenase biosynthesis [64], was up-regulated (Fig. 4). This result might be responsible for the observed decrease in the Chlorophyll a/b ratio in the mutants.

Fig. 4.

DEGs in MEP, MVA, and metabolism of carotenoids and chlorophylls. Green represents down-regulated genes, red represents up-regulated genes. The MEP, MVA, and metabolism of carotenoids and chlorophyll map are summarized according to previous reports [61, 62, 63]. DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; ISPS, isoprene synthase; FDPS, farnesyl diphosphate synthase; ACAT, acetyl-CoA C-acetyltransferase; HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; GCR, unclear receptor subfamily 3, group C, member 1; LUT1, Cytochrome P450 superfamily protein; ICYE, lycopene epsilon cyclase; CCS1, copper chaperone CCS1; NCED, putative 9-cis-epoxycarotenoid dioxygenase 3; ABA2, NAD(P)-binding Rossmann-fold superfamily protein; POR, cytochrome p450 oxidoreductase; SGR, AP2-domain transcription factor SGR; CLH2, chlorophyllase 2; HCAR, coenzyme F420 hydrogenase family / dehydrogenase, beta subunit family; CAO, chloroplast signal recognition particle component; HemB, porphobilinogen synthase; PGB, porphobilinogen; Hmb, hydroxymethylbiliin; UroⅢ, uroporphyrinogen Ⅲ; CoprogenⅢ, Coproporphyrin Ⅲ; Proto IX, protoporphyrin Ⅸ; Mg-Proto IX, Mg-Protoporphyrin IX.

The direct precursor of carotenoids biosynthesis is geranylgeranyl pyrophosphate (GGPP), which is also a precursor of chlorophyll biosynthesis [24]. Therefore, the DEGs of terpene metabolism were analysed in detail. The results indicated that HfDXS, which encodes a key enzyme in the MEP pathway, was significantly negatively regulated (Figs. 3,4). The expression level of HfDXS in albino mutant plants was down-regulated 3.5 times compared with that in the wild-type control (Supplementary Table 5). Regarding the mevalonate pathway (MVA) pathway for terpene metabolism, the genes encoding acetyl-CoA C-acetyltransferase and hydroxymethylglutaryl-CoA synthase were down-regulated (Fig. 4). These DEGs act upstream of the metabolic pathways of MEP and MVA. Therefore, changes in these DEGs likely substantially contributed to decreases in chlorophyll and carotenoid content in the leaves of the mutant plants. Regarding other genes relevant to carotenoid biosynthesis, two up-regulated and three down-regulated DEGs were found (Table 2). The up-regulated DEGs were genes involved in the biosynthesis of $ϵ$- carotene and lutein. The foldchange values were 2.40 and 8.62, respectively (Table 2). The down-regulated DEGs were genes involved in biosynthesis of aldehydes, capsaicin and 2-cis- 4-trans-xanthin, and the fold change range was 0.38, 0.43 and 0.19, respectively (Table 2). The transcription factor WRKY genes regulate the expression of DXS genes [65, 66]. Therefore, the bioinformatic analysis of the differential expression of the HfWRKY genes with RNA-seq was performed and followed by quantitative PCR confirmation, showing that HfWRKY24 was remarkably upregulated (Fig. 3 and Supplementary Table 5).

The photosynthetic pigment contents of Alb${}^{+/+}$ and Alb${}^{+/-}$ plants were measured after being identified by the albino phenotype that appeared in self-pollination-derived progeny. The content of chlorophyll and carotenoids in the leaves of Alb${}^{+/-}$ plants was significantly lower than that of the Alb${}^{+/+}$ plants (Supplementary Table 7). Measuring the photosynthetic characteristics of Alb${}^{-/-}$ was difficult because of the small plant size and short surviving time. The results showed that not only the net photosynthetic rate (Pn), but also the transpiration rate (Tr), intercellular CO${}_2$ (Ci), and water use efficiency (WUE) of Alb${}^{+/+}$ were significantly higher than those of the Alb${}^{+/-}$ plants (Fig. 5A–D). The photosynthetic response curves of Alb${}^{+/-}$ and Alb${}^{+/+}$ plants were further analysed because of the difference in Pn between the Alb${}^{+/-}$ and Alb${}^{+/+}$ plants. The Pn of the Alb${}^{+/-}$ plants was significantly lower than that of the Alb${}^{+/+}$ plants when the light intensity was higher than 100 µmol photons m${}^{-2}$$\cdot$s${}^{-1}$ (Fig. 5E), indicating that the light saturation point of Alb${}^{+/+}$ plants was higher than that of the Alb${}^{+/-}$ plants. qPCR was performed to confirm whether the above differences in phenotypes between Alb${}^{+/+}$ and Alb${}^{+/-}$ plants were accompanied by changes in the expression levels of HfDXS and HfWRKY24. The expression level of HfDXS in the Alb${}^{+/+}$ leaves was slightly higher than that in the Alb${}^{+/-}$ leaves, but the expression level of HfWRKY24 in the Alb${}^{+/+}$ leaves was significantly higher than that in the Alb${}^{+/-}$ leaves (Supplementary Table 8). In terms of photosynthetic characteristics, the Pn of Alb${}^{+/+}$ declined after drought treatment. However, the trend of decline slowed down after 4 d treatment. Meanwhile, the Pn of Alb${}^{+/-}$ decreased continuously during the treatment period. The change trend of the stomatal conductance was consistent with that of Pn between Alb${}^{+/+}$ and Alb${}^{+/-}$ after drought treatment (Fig. 6).

Fig. 5.

Comparison of the net photosynthetic capacity, and light response curve between Alb${}^{\mathrm{+}\mathbf{}\mathrm{/}\mathrm{+}}$ and Alb${}^{\mathrm{+}\mathbf{}\mathrm{/}\mathrm{-}}$ plants of daylily. (A–D) Photosynthetic capacity of Alb${}^{+/+}$ and Alb${}^{+/-}$ plants of daylily was compared under laboratory conditions. (E) Light response curve of Alb${}^{+/+}$ and Alb${}^{+/-}$ plants. Mean values $\pm$ SE (n = 3); Significant values *p 0.05, **p 0.01; ns, no significance.

Fig. 6.

Photosynthetic capacity and relative water content of Alb${}^{\mathrm{+}\mathbf{}\mathrm{/}\mathrm{+}}$ and Alb${}^{\mathrm{+}\mathbf{}\mathrm{/}\mathrm{-}}$ plants of daylily after drought treatment. (A–E) Photosynthetic capacity and relative water content of Alb${}^{+/+}$ and Alb${}^{+/-}$ plants of daylily were compared under progressive drought stress under laboratory conditions. Data were obtained from three two-year-old plants, and similar trends were observed in repeated experiments. (F) Soil moisture content during drought treatment. Mean values $\pm$ SE (n = 3); Significant values *p 0.05, **p 0.01; ns, no significance.

As photosynthetic efficiency affects the drought tolerance of plants [51], RWC of Alb${}^{+/+}$ and Alb${}^{+/-}$ plants were measured after drought treatment. The RWC of the leaves in Alb${}^{+/-}$ plants declined immediately after drought treatment. Meanwhile, the RWC of the leaves in Alb${}^{+/+}$ plants showed a significant reduction only after 1 week of treatment (Fig. 6), indicating that the Alb${}^{+/-}$ plants were more sensitive to drought conditions than the Alb${}^{+/+}$ plants. This result prompted us to investigate whether albino mutant seedlings were also sensitive to drought conditions. Owing to the short life of mutant seedlings cultured in soil, sterile culture with sucrose and PEG were used for drought stress treatment. Initially, the albino leaves grew similarly to the wild type (Fig. 7A), and as the number of days of stress increased, the albino leaves began to wilt and collapse (Fig. 7B). The mature leaves of albino seedlings almost completely wilted and were lodging after treatment (Fig. 7C), and dark brown spots appeared on the mature leaves (Fig. 7D). Meanwhile, the wild-type seedlings were almost unaffected by drought treatment (Fig. 7C). These results indicated that albino mutant is sensitive to drought, because high-MW PEG is considered the most suitable modelling system for modelling drought treatment in plants grown in vitro [67]. Furthermore, the 1-year-old plants cultured in pots with soil were exposed to drought conditions. The results showed that the leaves of Alb${}^{+/-}$ plants curled downward and wilted after 1 week of drought treatment (Fig. 7F–K). However, the Alb${}^{+/+}$ plants were almost unaffected after the first week of drought treatment (Fig. 7M–R). The leaves of Alb${}^{+/-}$ and Alb${}^{+/+}$ plants were smooth and flat before drought treatment (Fig. 7E,L). In addition, the degree of curling of Alb${}^{+/-}$ leaves was higher than that of the Alb${}^{+/+}$ leaves (Fig. 7K,R). These results indicated that Alb${}^{+/+}$ plants were more tolerant to drought treatment compared with Alb${}^{+/-}$ plants.

Fig. 7.

Sensitivity of albino mutant plants of daylily to drought conditions. Seedlings of self-pollinated progeny of daylily cultivar ‘black-eyed stella’ were transplanted to a medium containing 10% PEG2000 after culturing on a PEG free medium for 2 weeks. (A) Seedlings before PEG-treatment. (B,C) Seedlings being treated for 3 and 6 days, respectively. (D) The albino leaves showing brown speckles after PEG-treatment. (E–R) One-year-old plants, grown in pot with soil, were treated with drought. (E,L) Potted Alb${}^{+/-}$ and Alb${}^{+/+}$ plants before drought-treatment. (F–J) Alb${}^{+/-}$ plants exposed to drought for 3 d, 6 d, 9 d, 12 d, 15 d, respectively. (M–Q) Alb${}^{+/+}$ plants exposed to drought for 3 d, 6 d, 9 d, 12 d, 15 d, respectively. (K) and (R) Leaves of potted Alb${}^{+/-}$ and Alb${}^{+/+}$ plants after drought treatment for 15 days.