Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice

Accepted Manuscript Title: Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice Author: Fei Zhang Pan Zhang Yu Zhang Shouchuang Wang Lianghuan Qu Xianqing Liu Jie Luo PII: DOI: Reference:

S0168-9452(16)30136-4 http://dx.doi.org/doi:10.1016/j.plantsci.2016.06.017 PSL 9442

To appear in:

Plant Science

Received date: Revised date: Accepted date:

17-3-2016 23-6-2016 24-6-2016

Please cite this article as: Fei Zhang, Pan Zhang, Yu Zhang, Shouchuang Wang, Lianghuan Qu, Xianqing Liu, Jie Luo, Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice, Plant Science http://dx.doi.org/10.1016/j.plantsci.2016.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice

Fei Zhanga,1, Pan Zhanga,1, Yu Zhanga, Shouchuang Wanga, Lianghuan Qua,b Xianqing Liub, Jie Luoa,*

a National

Key Laboratory of Crop Genetic Improvement and National Center

of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China b

College of Life Science and Technology, Huazhong Agricultural University,

Wuhan 430070, China 1These

two authors contributed equally to the work.

*Correspondence: [email protected];

1

Highlights We identified a rice Chl-deficient mutant caused by mutants in OsAld-Y which encodes a fructose-1,6-bisphosphate aldolase. Reduced expression of OsAld-Y affected the accumulation of sugar intermediates. Subcellular localization analysis demonstrated that OsAld-Y was localized to peroxisome which provides a novel insight into the mechanism underlying Chl biosynthesis.

Abstract Chlorophyll

plays

remarkable

and

critical

roles

in

photosynthetic

light-harvesting, energy transduction and plant development. In this study, we identified a rice Chl-deficient mutant, ygdl-1 (yellow green and droopy leaf-1), which showed yellow-green leaves throughout plant development with decreased content of Chls and carotene and an increased Chl a/b ratio. The ygdl-1 mutant also exhibited severe defects in chloroplast development, including disorganized grana stacks. Sequence analysis revealed that the mutant

contained

a

T-DNA

insertion

within

the

promoter

of

a

fructose-1,6-bisphosphate aldolase (OsAld-Y), which dramatically reduced the OsAld-Y

mRNA level,

and

its

identity

was

verified

by

transgenic

complementation. Real-time PCR analysis showed that the expression levels of

genes

associated

with

chlorophyll

biosynthesis

and

chloroplast

development were concurrently altered in the ygdl-1 mutant. The expression of 2

OsAld-Y-GFP fusion protein in tobacco epidermal cells showed that OsAld-Y was localized to the peroxisome. In addition, the analysis of primary carbon metabolites revealed the significantly reduced levels of sucrose and fructose in the mutant leaves, while the glucose content was similar to wild-type plants. Our results suggest that the OsAld-Y participates in Chl accumulation, chloroplast development and plant growth by influencing the photosynthetic rate of leaves and the sugar metabolism of rice.

Abbreviations: FBA: fructose-1,6-bisphosphate aldolase; Chl: chlorophyll; WT, wild-type Cald: the complemented line; FBPase: fructose-1,6-bisphosphatase; ygdl-1: yellow green and droopy leaf-1; PARi: photosynthetically active radiation intensity.

Keywords:

fructose-1,6-bisphosphate

aldolase;

chlorophyll;

sugar

metabolism; peroxisome

1. Introduction Chlorophyll (Chl), one of the most common organic molecules on earth, exists ubiquitously in nature in photosynthetic organisms, such as green plants and cyanobacteria. In addition to supplying the green color in plants and cyanobacteria, Chl molecules are the primary photoreceptor pigments that absorb light energy and drive electron transfer in the reaction center of the 3

photosynthetic system to synthesize carbohydrates from CO2 and water [1]. Chl consists of two moieties, chlorophyllide and phytol, which are formed via the tetrapyrrole or the isoprenoid biosynthetic pathways respectively. Mutations in the Chl biosynthetic pathway genes affect leaf color and are considered to be ideal materials to explore the molecular mechanism of Chl biosynthesis and chloroplast development [2-5]. For instance, disruption of the Chl synthase encoding gene (YGL1) leads to decreased Chl contents and delayed chloroplast development, resulting in a yellow-green leaf phenotype in young plants in rice [2]. Chl content in plants can be influenced by both external and internal factors such as light, salt, osmotic stress and leaf age due to alterations in the genes expression or post-translational modification of proteins involved in Chl synthesis and Chl degradation [6-10]. For example, a missense mutation of the fructose-1,6-bisphosphatase (FBPase) encoding gene results in a yellow-green leaf phenotype and severe growth retardation in rice [11]. It has long been demonstrated that the disruption of chloroplast function has a profound effect on chlorophyll biosynthesis and stability [6, 8]. In addition to its role in photosynthesis, chloroplast is also involved in the biosynthesis of hormones, sugars and amino acids. Some energy metabolism processes occur not only in chloroplasts but also in other two types of organelles, the peroxisomes and mitochondria. These organelles work cooperatively in plant energy metabolism processes such as photorespiration. Furthermore, most of 4

the proteins that participate in energy metabolism and chlorophyll biogenesis are found in both chloroplasts and mitochondria. However, only a few peroxisome proteins are also implicated in chlorophyll biogenesis. For example, Peroxisomal Biogenesis Factor 2 (PEX2), a peroxisome protein involved in peroxisome assembly and matrix protein import, is required for photomorphogenesis

in

Arabidopsis

[12].

The

mutation

of

SNOWY

COTYLEDON 3 (sco3), localized to the periphery of peroxisomes, resulted in reduced chlorophyll accumulation and defective chloroplast development, possibly through interaction with the cytoskeleton [13]. Here, we identified in rice an ygdl-1 (yellow green and droopy leaf 1) mutant containing a T-DNA insertion in the promoter region of a gene encoding FBA, referred to as OsAld-Y. The mutant exhibited a yellow-green leaf phenotype, decreased levels of Chl, and abnormal chloroplasts throughout plant development. Subcellular localization analysis demonstrated that OsAld-Y was localized to the peroxisome and implicated in the sugar metabolism in Chl biosynthesis. Our results provide a novel insight into the mechanism underlying Chl biosynthesis and chloroplast development in rice.

2. Materials and Methods 2.1 Plant materials

The mutant line 04Z11EN05 (ygdl-1; rice (Oryza sativa ssp japonica cv Zhonghua

11)

was

screened

from 5

the

RMD

database

[14,

15]

(http://rmd.ncpgr.cn/). All mutants and transgenic plants were planted in an experimental field at Huazhong Agricultural University (Wuhan, China) in summer and in a greenhouse during the winter.

2.2 Vector construction and plant transformation

To prepare the complementation vector, a 4.4-kb genomic DNA fragment containing the 2297-bp upstream sequence and 2155-bp coding region of OsAld-Y

was

isolated

by

digestion

of

the

Clemson

BAC

clone

OSJNBa0018M24 (kindly provided by R. Wing, University of Arizona) and inserted

into

the

binary

vector

pCAMBIA2301.

The

empty

vector

pCAMBIA2301 was used as a negative control. The transformation recipient was a callus culture induced from seeds of homozygous for ygdl-1. To fuse the OsAld-Y (Os06g40640) promoter to the GUS gene, the promoter of OsAld-Y, a 1605-bp fragment upstream of the start Condon ATG, was amplified. The PCR product was cloned into pDONR207 by BP recombination. After

sequencing,

the

correct

clone

was

introduced

into

the

Gateway-compatible GUS fusion vector pGWB3 to produce OsAld-YpGUS. The constructs were then introduced into wild-type Zhonghua 11 callus, with the empty vector as a control. All primers for genotyping and vector construction are listed in Supplementary Table S1.

6

2.3 GUS staining

GUS staining was performed as previously described [16]. Samples were transferred to X-gluc buffer overnight at 37°C, fixed in formalin/ ethanol/acetic acid fixation solution (3.7% formaldehyde, 50% ethanol, 5% acetic acid) at 4°C for 16 h, and washed with 75% ethanol overnight to remove the chlorophyll. The cleared samples were observed under a stereo light microscope (Leica MZFL III), and images were taken using a digital camera (Canon 60D).

2.4 RT-PCR and quantitative real-time PCR

RNA was extracted using an RNA extraction kit (TRIzol reagent; Invitrogen) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using 5 μg of RNA and reverse-transcribed using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Quantitative Real-time PCR was performed in a 96-well plate in an ABI Step One Plus PCR system (Applied Biosystems) using SYBR Premix reagent F-415 (Thermo Scientific). The expression levels were measured using the relative quantification method [17]. The primers for RT-PCR and quantitative RT-PCR are listed in Supplementary Table S1.

2.5 Photosynthetic rate measurement

Rice plants were grown in a steady-state greenhouse for two-months. Measurements were taken on leaves exposed to a saturating photosynthetic 7

photon flux density of 1500 μmolm−2s−1 actinic white light at 28°C, with the relative humidity when the leaf cuvette ranging between 45% and 55% (to avoid midday depression in photosynthesis). Before the measurements, the leaves were fully adapted to dark conditions for 30 min. The plants were measured using a portable infrared gas analyzer (Model L1-6400XT, Li-Cor, Lincoln, NE, USA). The leaf chamber was the open type, and measurements were performed at 8:00-12:00am each day for WT, mutant and complemented line plants. The photosynthetically active radiation intensity (PARi) levels during measurement were 0, 50, 100, 200, 400, 800, 1000, 1500, 2000 μmolm−2s−1, with the leaf temperature was 28°C and the CO2 concentration 400 ppm [18]. The observations were recorded after the plant reached steady-state photosynthesis. Maximum net photosynthetic rate (Pnmax), Dark respiration (Rd) and Light compensation point (LCP) were obtained by analyzing the photosynthetic light-response curve using MATLAB (Matrix Laboratory) [19]. All measurements were recorded three times and averaged on each occasion of sampling.

2.6 Sequence alignment and phylogenetic tree construction

The deduced amino acid sequence of the OsAld-Y gene was aligned with previously reported FBA genes from rice and other plant species using the ClustalW program [20]. A phylogenetic tree was constructed using the MEGA software version 4.0 via the neighbor-joining method [21, 22]. 8

2.7 Subcellular localization of OsAld-Y

To examine the subcellular localization of OsAld-Y in rice, the entire open reading frame without the stop codon was amplified by PCR. The amplified product was cloned into pDONR207. After sequencing, the correct entry clone was individually introduced into the Gateway-compatible GFP fusion vector pB7WGF2, generating the vector 35S::OsAld-Y-GFP plasmid, which was applied to tobacco leaves through Agrobacterium-mediated transformations. Leaves from the 35S::OsAld-Y-GFP transgenic tobacco plants were examined using a confocal laser scanning microscope (Leica TCS SP2). The plasmid RFP-OsOPR7 was used as a peroxisome targeting control. At least 10 separate samples were observed, and a representative image is presented.

2.8 Measurement of chlorophyll content

Two-week-old seedings grown in MS media and plants grown in soil for 6 weeks were used to test the chlorophyll content. Chlorophyll was extracted with 80% acetone from fresh leaves, whose chlorophyll content was then determined according to the method of Porra, Thompson & Kriedemann [23].

2.9 Enzyme assay for fructose1,6-bisphosphate aldolase

The enzyme assay was conducted using The ALD Continuous loop response spectral method of quantitative detection kit for plants (GenMed Scientifics Inc. U.S.A) according to the user manual. Briefly, the aldolase 9

reaction, buffered in tris acetate EDTA (TAE) and ethylenediaminetetraacetic acid (EDTA), was coupled with triosephosphate isomerase and glycerol 3-phosphate dehydrogenase reactions and assayed at 25°C by monitoring NADH utilization at 340 nm [4, 11, 24]. The assay was repeated three times for each line, and mean values are indicated with estimated error bars.

2.10 Transmission electron microscopy

Wild-type and ygdl-1 mutant leaf samples were harvested from 1-month-old plants grown in the experimental field. Leaf sections were fixed in a solution of 2% glutaraldehyde and further fixed in 1% OsO4. Tissues were stained with uranyl acetate, dehydrated in ethanol, and embedded in Spurr’s medium prior to thin sectioning. The samples were stained again and examined with a JEOL 100 CX electron microscope.

2.11 Quantitative analysis of sugar content

Leaf material was harvested from 5-week-old plants grown in soil and approximately 100 mg of frozen leaf was used for analysis. Soluble sugar was extracted using a fixation solution (225 μl of methanol, 120 μl of CHCl3, and 240 μl of ddH2O) at 70°C for 15 min. Samples were centrifuged at 12000 rpm for 10 min; 200 μl of supernatant was transferred to a new tube and then dried at 80°C. For methoximation, 40 μl of methoxyamine hydrochloride in pyridine (20

mg/ml)

was

added

for

90

min

10

at

30°C.

Then,

60

μl

of

N-methyl-N-trimethylsilyltrifluoroacetamide was added, and the mixture was incubated at 37°C for 30 min. The derivatives were analyzed by gas chromatography-mass spectrometry on a Thermo DSQII mass spectrometer using a DB-5ms column. A temperature program was then implemented as follows: initial temperature 70°C, followed by heating to 300°C, which was held for a further 3 min. Myo-inositol was used as an internal standard.

2.12 Measurements of the maximal quantum yield of photosystem II (PSII)

The maximal quantum yield of PSII (Fv/Fm) was determined by chlorophyll a fluorescence measurements. Measurements were performed in greenhouse in the morning (9:00–10:00am). The fluorescence yield was measured on the flag leaves of potted rice using a Handy PEA instrument (Hansatech Instruments, King’s Lynn, UK). The maximal and minimal fluorescence intensity of dark-adapted leaves (Fm and Fo, respectively) were determined after 30min dark adaptation using leaf clips. The intensity and duration of the saturation pulse applied to determine Fm were 3500 mmol photons m-2 s-1 and 1s, respectively. The Fv/Fm was calculated as Fv/Fm = (Fm – Fo)/Fm [25, 26].

3. Results 3.1 Characterization of a chlorophyll–deficient rice mutant

A yellow-green and droopy leaf mutant (RMD_04Z11EN05), designated as ygdl-1, was isolated from the T-DNA insertion mutant library generated by the 11

transforming rice japonica variety Zhonghua 11 with an enhancer-trap construct [14, 15]. This mutant displayed yellow-green leaves from the four-leaf stage under standard greenhouse conditions (Fig. 1A, B). The same phenotype was observed in mature plants grown in paddy fields. To characterize the yellow leaf phenotype of ygdl-1, the chlorophyll content was measured. The results showed that the content of Chl a, Chl b and carotene in the ygdl-1 mutant was 56.4%, 43.9%, and 70.2% of their values in wild-type plants, respectively (Table 1) Electron microscope analyses revealed that grana and its stacks in the ygdl-1 mutant appeared less dense (Fig. 1C, E) and lacked grana membranes compared with the wild-type. The thylakoid membrane systems of the chloroplasts were disturbed in the ygdl-1 mutant, and the membrane spacing was not as clear as in wild-type chloroplasts (Fig. 1D, F). Together, these results revealed that the development of the chloroplast thylakoid was disrupted in the ygdl-1 mutant.

3.2 Loss of ygdl-1 function decreases Chl content

The ygdl-1 mutant harbored a T-DNA insertion approximately 230 bp upstream of the translation start site ATG of the gene encoding a fructose-1,6-diphosphate aldolase isoenzyme (Fig. S1, S2), named OsAld-Y (Fig. 2A). PCR analyses revealed that the T-DNA insertion cosegregated with the phenotype in the T2 generation (Fig. 2B). All plants homozygous for the

12

T-DNA insertion displayed the mutation phenotype, while the heterozygotes and plants without the T-DNA insertion showed the wild-type phenotype. Furthermore, real-time PCR analysis showed that the endogenous OsAld-Y transcript was greatly reduced in the T-DNA insertion homozygous plants (Fig. 2C). We then measured the total FBA activities for the WT, ygdl-1, and Cald lines. The results showed that the total FBA activity in ygdl-1 decreased by 50% compared with the WT cells (Fig. 2D). In addition, the total FBA activity in Cald was restored to the WT level, which is consistent with the results obtained from the gene expression analysis (Fig. 2C). These results indicate that ygdl-1 is a knock-down mutant of OsAld-Y that impairs FBA function. To further confirm that the T-DNA insertion in OsAld-Y gene is responsible for the ygdl-1 phenotype, we transformed the mutant line with a binary plasmid carrying an approximately 4.4 kb wild-type genomic fragment containing the 2297 bp upstream sequence and 2155 bp coding region of OsAld-Y. Real-time PCR analysis indicated that the complemented lines expressed OsAld-Y at similar levels to the wild-type (Fig. 2E). As expected, the color of the leaves and the levels of Chl a and Chl b were all restored to the levels of wild-type plants upon transformation with the OsAld-Y gene (Fig. 2F, G). These results confirmed that the observed abnormal phenotypes of the ygdl-1 mutant plants resulted from the mutation of the OsAld-Y gene.

13

3.3 OsAld-Y affects the photosynthetic rate and Chl biosynthesis and chloroplast development genes associated with the photosynthetic rate of rice

The expression of certain genes required for Chl biosynthesis, which encode chlorophyllide a oxygenase (CAO1), glutamyl-tRNA reductase (HEMA1) and NADPH-protochlorophyllide

oxydoreductase

(POR4),

was

significantly

down-regulated in the ygdl-1 mutant compared with wild-type (Fig. 3A). Similarly, the expression levels of chloroplast development-related genes Cab1R and Cab2R, which encode two light-harvesting Chl a/b-binding proteins of PSII [27], and psaA and psbA, which separately encode two reaction center polypeptides of PSI and PSII, were also significantly down-regulated (Fig. 3A). We then measured the photosynthetic rates in the leaves of the WT, ygdl-1, and Cald lines, and the results showed that the photosynthetic rate of the ygdl-1 mutant was significantly lower than WT, while similar levels were observed for the WT and Cald plants (Fig. 3B). Maximum net photosynthetic rate (Pnmax), Dark respiration (Rd) and Light compensation point (LCP) were obtained by analyzing the photosynthetic light-response curve using MATLAB. We found that the Pnmax and Rd were significantly reduced in the ygdl-1 mutants than in WT plants, while no significant difference was observed for LCP. The Cald lines could partly recover the mutant phenotype in terms of their Pnmax and Rd values (Table 2). And the maximal quantum yield of PSII (Fv/Fm) was also decreased in ygdl-1 mutant (Fig. S5). Results from the analyses of cell structure (Fig. 1C-F), gene expression (Fig. 3A), and the 14

photosynthetic parameters (Fig. 3B, Table 2, and Fig. S5) all indicate that photosystem II (PSII) efficiency was decreased in ygdl-1 mutants than in the WT plants. Together, these results suggest that OsAld-Y is involved in determining the Chl content, chloroplast development and photosynthetic rate of rice.

3.4 Expression analysis of OsAld-Y

To investigate the expression profile of OsAld-Y, we searched the CREP rice gene expression database (http://crep.ncpgr.cn) [28]. We observed that OsAld-Y was widely expressed in a number of tissues, including the leaf, sheath, culm, hull, stamen and endosperm, with relatively lower levels in the root and young panicle (Fig. S3). Consistent with the results of the microarray experiments, notably higher expression levels of OsAld-Y were detected in photosynthetic organs, including the leaf, culm and sheath, throughout the stages of plant development (Fig. 4A). To further examine the spatiotemporal expression profile, a construct was produced in which the OsAld-Y promoter (approximately 2.1 kb upstream of the translation start site) was fused to the β-glucuronidase gene (GUS) to transform wild-type plants. GUS activity was detected in the leaf, sheath and stem, as well as non-photosynthetic organs, including the hull, stamen and ovary, which was consistent with the expression pattern observed by real-time PCR analysis (Fig. 4B-G). The preferential expression of OsAld-Y in

15

autotrophic source tissues strongly suggested that OsAld-Y functions mainly in photosynthetic source tissue, although the expression of this gene was also detected in non-photosynthetic organs.

3.5 Effects of the mutation on yield parameters

In addition to the leaf color, most of the agronomic traits were changed significantly in ygdl-1 mutant compared with the wild-type, including reduced plant height and decreased panicle length and grain width, as well as reduced 1,000-grain weight. However, no agronomical defects could be observed for Cald plants compared with their WT counterparts (Fig. 5A-D). These data suggested that the mutation of OsAld-Y had detrimental effects on the yield parameters of rice.

3.6 Reduced expression of OsAld-Y affected the accumulation of sugar intermediates

Analyses of the soluble sugar content in the leaves of 6-week-old plants revealed that the sucrose and fructose content in the ygdl-1 mutant was 30% and 70% lower than in WT plants, respectively (Fig. 6A, C). We then examined the expression of two key genes in the sucrose biosynthesis pathway that encode a cytosolic fructose-1,6-bisphosphatase and a sucrose phosphate synthase. As shown in Figure 6D, the cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase genes were strongly down-regulated in the

16

ygdl-1 mutant compared with the wild-type. These results indicated that a shortage of sucrose occurs in the ygdl-1 mutants. To investigate whether exogenous feeding with sucrose would rescue the pale-green-leaf phenotype of the ygdl-1 mutants. The rice seeds were germinated and then grown in medium with (+) or without (-) sucrose for approximately three weeks. Then, three-week-old seedlings were further treated with darkness for 72 h, and the flag leaves were used to measure chlorophyll content. After dark treatment, the ygdl-1 mutants showed reduced chlorophyll accumulation when grown in sucrose-deficient medium (Fig. 6E). However, this phenotype was rescued by growing them in medium supplemented with 5% sucrose (Fig. 6E). Together, these results are central to the suggestion that low sucrose levels resulted in chlorophyll deficiency in the ygdl-1 mutant.

3.7 OsAld-Y is localized to the peroxisome

To determine the subcellular localization of the OsAld-Y protein, a translation fusion between the full-length OsAld-Y coding region and the cDNA for the green fluorescent protein driven by the 35S cauliflower mosaic virus promoter was constructed and introduced into tobacco epidermal cells by micro-bombardment, then visualized using a confocal laser microscope. OsAld-Y::eGFP showed a punctate pattern of green fluorescence, which colocalized with the peroxisome enzyme OsOPR7 (12-oxo-phytodienoic acid 17

reductase) (Fig. 7C-F), indicating that OsAld-Y is localized to the peroxisome.

4. Discussion Chl-deficient mutants are ideal genetic material for the study of the molecular mechanisms that regulate Chl biosynthesis and chloroplast development in plants. Although a number of such mutants have been discovered [3, 29-32], Chl-deficient phenotypes caused by mutations in fructose-1,6-bisphosphate aldolase (FBA) homologs have not been previously reported in rice. In this study, we identified and functionally characterized a new chlorophyll-deficient mutant, ygdl-1, containing a T-DNA insertion within the OsAld-Y promoter that dramatically reduced the OsAld-Y mRNA level. The ygdl-1 mutant exhibit a yellow-green leaf phenotype throughout plant development and is quite different from most Chl-deficient rice mutants, which exhibit abnormal leaf coloration at the seedling stage but are restored in later stages [33]. Chl is absolutely necessary for the building of photosynthetic reaction centers and light-harvesting complexes, and an appropriate Chl a/b ratio is important in the control of photosynthetic antenna size [34]. A decrease in the Chl content and an increase in the ratio of Chl a/b were observed in the ygdl-1 mutant (Table 1), indicating that the total number of photosystems decreased and there might be fewer light-harvesting antenna complexes than in wild-type, similar to another rice Chl-deficient mutant [3]. This proposal is consistent with the decreased mRNA level of CAO1 (Fig. 3A), which is 18

responsible for the conversion of Chl a to Chl b and for the increased Chl a/b ratio in ygdl-1 plants. Chloroplast development relies not only on the coordinated synthesis of Chl and thylakoid proteins but also on their assembly into chloroplast thylakoids. The decrease of Chl synthesis and abnormal ratio of Chl a/b may disrupt the thylakoid ultrastructure, reducing the assembly of light-harvesting complexes in the thylakoid membrane. Transmission electron micrography demonstrated that the yellow-green leaves of ygdl-1 plants exhibited abnormal chloroplast morphologies (Fig. 2E, G). Furthermore, the abnormal pigment content and chloroplast structure in the ygdl-1 mutant may contribute to the partial repression of Chl synthesis. Two main isoforms of fructose-1,6-bisphosphate aldolase (FBA) exist in plants, one located in the cytosol and the other in chloroplasts [35], and different characteristics could be found [36]. Chloroplast FBA is an essential enzyme in the Calvin cycle, in which it produces metabolites for starch biosynthesis [4, 37]. We have carried out RT-PCR analyses of the Calvin cycle enzymes and the results are shown in Fig. S4. Expressions of genes required for Calvin cycle, encoding small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCS), large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

(RBCL),

ribulose-1,5-bisphosphate

carboxylase/oxygenase activase (RCA), sedoheptulose-1,7-bisphosphatase (SBP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [38], were 19

significantly up-regulated in ygdl-1 mutant compared with wild-type (Fig. S4), which is in line with previous studies [39, 40]. We infer that there might be a mechanism of increasing the expression of the Calcin cycle enzymes to compensate the decrease of the aldolase activity in the mutants, although further research is necessary to support this hypothesis.

The cytosol FBA is a part of the sucrose biosynthetic pathway and a key metabolic enzyme involved in the glycolytic/gluconeogenesis pathways in plants [41, 42], which can lead to the accumulation of water-soluble carbohydrates and the synthesis of ATP [43]. Both isoforms play important roles in the carbohydrate metabolism and in the production of triose phosphates and their derivatives in signal transduction [24]. Recently, Liu et al. [36] reported rice OsNOA1/RIF1 RNAi mutant seedlings exhibiting chlorosis, with reduced pigment content and lower photosystem II (PSII) efficiency. In this RNAi mutant, the up-regulation of genes encoding certain classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, including OsFBPs (two isoforms), was observed. By contrast, the OsAld-Y gene was down-regulated. Reduced abundances of enzymes in photorespiration, gluconeogenesis, and amino acid metabolism were also detected in the RNAi mutant. Unlike the other two FBPs functioning in the glycolytic pathway, OsAld-Y may participate in the gluconeogenesis pathway. In accordance with this concept, we also observed moderate expression of OsAld-Y in the non-photosynthetic tissues of rice. 20

Here, our results showed that the content of sucrose and fructose was clearly reduced in the ygdl-1 mutant compared with the wild-type and restored in the complemented lines (Fig. 6A-C), in parallel with the down-regulation of fructose-1,6-bisphosphatase (FBPase) and sucrose phosphate synthase (SPS1), which are critical regulatory enzymes in gluconeogenesis, detected in the mutant (Fig. 6D). These results are strong indications of the important role of OsAld-Y in the gluconeogenesis pathways and the accumulation of water-soluble carbohydrates in rice. Thus, it is proposed that the depletion of carbon resources in the mutant would eventually interfere with Chl biosynthesis. This contention is further supported by the fact that the ygdl-1 mutant does not cause chlorophyll loss when the mutant plant is supplied with exogenous sugar (Fig. 6E). In this study, the transient expression of GFP::OsAld-Y showed that OsAld-Y was located in the peroxisome, despite lacking any recognizable Nor C-terminal peroxisome targeting signals (PTSs) (Fig. 7A-D). Notably, proteins without obvious PTSs have also been identified in the leaf peroxisome proteome. These proteins may be localized by internally located PTS peptides, by alternative targeting signals or pathways, by “piggy-backing” or by peripheral and integral membrane proteins [44, 45]. For example, a key enzyme of the NAD+ salvage pathway, Pnc1p, a nicotinamidase with no functional terminal PTS, is co-imported into peroxisomes by piggyback transport via the glycerol-producing PTS2 protein glycerol-3-phosphate 21

dehydrogenase Gpd1p [46]. Further research is needed to determine how OsAld-Y is transported. Peroxisomes are small and dynamic single membrane-bound organelles found in nearly all eukaryotic cells that function as essential crosstalk in certain metabolic networks within the cell through physical and metabolic links with other cellular compartments, such as chloroplasts and mitochondria [47]. Plant peroxisomes play critical roles in the β-oxidation of fatty acids and hydrogen peroxide (H2O2) catabolism. They also mediate pathways such as the glyoxylate cycle and photorespiration, as well as contributing to pathogen defense and to developmental processes such as embryogenesis and photomorphogenesis [22, 48-51]. Here, we found that OsAld-Y is present in the peroxisome, raising the possibility that the glycolytic pathway is associated with the peroxisome, possibly to ensure the delivery of the glycolytic intermediate pyruvate to the site of the glyoxylate cycle, or to transport ATP generated from the glycolytic pathway to the peroxisome during plant growth. A similar study determined the association of glycolysis enzymes with the mitochondrion in Arabidopsis [24, 31]. Although the precise mechanism is unknown, the Chl-deficient phenotype in ygdl-1 suggested that certain signals or intermediates generated in the peroxisome can regulate chloroplast biogenesis or Chl biosynthesis. Further research is needed to determine how the expression of OsAld-Y is regulated and how the peroxisome and nucleus are coordinated to regulate the Chl metabolism and chloroplast development. 22

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31070267), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB127001), the National High Technology R&D Program of China (863 Program) (No. 2012AA10A303), and the Program from

Fundamental Research Funds for the Central Universities

(No.

2662015PY196). We would like to thank Dr. Yong Li from College of Plant Science and Technology, Huazhong Agricultural University for the assistance in the photosynthetic rate and maximal quantum yield of photosystem II (PSII) measurements.

Supplementary Data Supplementary data associated with this article can be found in the online version.

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31

Figure Captions Fig 1. Phenotype of the rice ygdl-1 mutant.

(A-B) Wild-type (left) and ygdl-1 mutant (right) plants (A) and leaves (B) at the tillering stage. (C–F) Electron microscopic analysis of the mesophyll cells of wild-type (C) and ygdl-1 mutant (D) leaves, and the chloroplast structure of the wild-type (E) and ygdl-1 mutant (F). g, grana stack; sl, stroma lamellae. Bar=5 cm in (A).

Fig 2. Loss function of ygdl-1 decreases Chl content. (A) OsAld-Y gene structure: T-DNA was inserted into the 5’-UTR region of OsAld-Y approximately 230 bp upstream of the translation start site (ATG). P1, P2 and P3 indicate primers used for genotyping. (B) PCR results for genotyping. W, H and M indicate wild-type plants, 32

heterozygotes and plants homozygous for the T-DNA insertion, respectively. (C) Real-time PCR showed that the expression level of OsAld-Y was greatly reduced in the ygdl-1 mutant. (D) Total FBP activity measurement of WT, ygdl-1 and Cald lines after 5 and 10 min reactions in vegetable cells. (E) Comparison of the wild-type (left), ygdl-1 (middle), and complemented lines (right) after heading, grown in a rice paddy field. (F) Comparison of a flag leaf from each of the plants shown in (E). (G) Chlorophyll content measurement of wild-type (WT), ygdl-1 and complemented lines. WT: wild-type; Cald: complemented line. Each data point represents the mean (±SD) from three separate experiments. Bar=10 cm in (D). Asterisks indicate values significantly different from the wild-type or control based on Student’s t test (*P < 0.05; **P < 0.01).

33

Fig 3. The effect of ygdl-1 on photosynthetic rate is associated with Chl biosynthesis and chloroplast development genes.

(A) Expression level of genes associated with chlorophyll biosynthesis, chloroplast development and photosynthesis assayed using real-time PCR in WT, ygdl-1 and Cald at heading stage. (B) Comparison of net photosynthetic rates among WT, ygdl-1 and Cald under different PARi in top second leaf. WT: wild-type; Cald: complemented line; Pn: net photosynthetic rate; PARi: photosynthetically active radiation. Each data point represents the mean (±SD) from three separate experiments. Asterisks indicate values significantly different from the wild-type control based on Student’s t test (*P < 0.05; **P < 0.01, ***P < 0.001).

34

Fig 4. Expression pattern of OsAld-Y.

(A) Expression analyses of OsAld-Y by real-time PCR. Three-leaf stage, leaf and shoot (z1); seeding with 2 tillers stage, leaf (z2); vegetative stage, leaf (z3); primordium differentiation stage, leaf (z4); 4-5 cm young panicle: leaf (z5), sheath (z6) and young panicle (z7); 5 days before heading (booting stage): flag leaf (z8), culm (z9), young panicle (z10); heading stage: flag leaf (z11), panicle (z12), stem (z13); one day before flowering, panicle (z14); 3 days after pollination, panicle (z15); 7 days after pollination: flag leaf (z16), culm (z17) and spikelet (z18); 14 days after pollination: culm (z19) and spikelet (z20). (B-G) GUS staining analysis of various tissues in the OsAld-Y pro:GUS transgenic lines at the flowering stage. GUS activity was detected in the flag leaf (B), sheath (C), stem (D), hull (E), stamen (F) and ovary (G). Each data point represents the mean (±SD) from three separate experiments.

35

Fig 5. Agronomic traits of the wild-type, the ygdl-1 mutant and the complemented lines.

Comparison of the plant height (A), panicle length (B), weight of 1,000 grains (C) and grain width (D) in the wild-type, ygdl-1 mutant, and complemented lines after heading, grown in the rice paddy field. Each data point represents the mean (±SD) from three separate experiments. Bar=2 cm in (A, B and D).

36

Fig 6. Soluble sugar content in ygdl-1 plants and exogenous sugar treatment in ygdl-1 mutant.

The amounts of sucrose (A), glucose (B), fructose (C) in the mature leaves of 6-week-old plants are shown. (D) Expression levels of genes associated with sucrose biosynthesis assayed using real-time PCR in the wild-type and ygdl-1 mutant at the heading stage. (E) Chlorophyll content of the wild-type and ygdl-1 mutant grown with (+) or without (-) sucrose supplementation. The chlorophyll content in the mutant is recovered by treatment with exogenous sucrose. Three-week-old rice plants grown in medium and with 3 d dark treatment were analyzed. Each data point represents the mean (±SD) from three separate experiments. (FW: fresh weight). 37

Fig 7. Subcellular localization of OsAld-Y::eGFP.

(A-D) Subcellular localization of OsAld-Y. OsAld-Y::eGFP (A) and a peroxisomal marker RFP::OsOPR7 (B) displayed strong co-localization (D) in tobacco epidermal cells (C). Bars=50 µm (A-D).

38

39

Table Table 1. Pigment content in the leaves of wild-type and ygdl-1 mutant in mg g-1 fresh weighta

Genotype

Chl a

Chl b

Carotene

Chl a/b

Wild-type

1.41±0.010

0.64±0.051

0.37±0.017

2.20±0.041

ygdl-1

0.80±0.040***

0.28±0.011***

0. 26±0.012**

2.83±0.056**

a Chlorophyll

(Chl) and carotene were measured in acetone extracts from the

flag leaf of rice at the tillering stage. The values shown are the mean (±SD) from three independent determinations. Asterisks indicate values significantly different from the wild-type or control based on Student’s t test (**P < 0.01; ***P < 0.001).

40

Table 2. The main photosynthetic parameters in leaves of wild-type, Cald line and ygdl-1 mutant plantsa

Genotype

Pnmax (μmol CO2m-2s-1)

Rd (μmol CO2m-2s-1) LCP(μmol m-2s-1)

Wild-type

23.80±1.23

-19.05±0.29

37.08±3.33

Cald

21.62±0.14

-17.64±1.29

37.90±2.91

ygdl-1

12.88±0.40 ***

-12.00±1.35 **

41.09±2.93

a

Maximum net photosynthetic rate (Pnmax), Dark respiration (Rd) and Light

compensation point (LCP) were obtained by analyzing the photosynthetic light-response curve using MATLAB. The values shown are the mean (±SD) from three independent determinations. Asterisks indicate values significantly different from the wild-type or control based on Student’s t test (**P < 0.01; ***P < 0.001).

41