Genome-wide transcriptional analysis of maize endosperm in response to ae wx double mutations

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GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 749762

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Genome-wide transcriptional analysis of maize endosperm in response to ae wx double mutations Xiang Li, Guang Hui Chen, Wei Yang Zhang, Xiansheng Zhang * State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian 271018, China Received for publication 3 February 2010; revised 25 August 2010; accepted 14 September 2010

Abstract Starch biosynthesis is important during endosperm development. Much has been known for the regulation of gene expression involved in starch synthesis, less information is available on the genome-wide expression profiles as a consequence of impaired starch synthesis. In this study, we examined the transcriptional responses through microarray analysis in an ae wx double-mutant with loss-of-function starch branching enzyme IIb (SBEIIb) and granule-bound starch synthase I (GBSSI). Through Gene Ontology enrichment analysis, we identified differentially expressed genes (DEGs) involved in chromatin organization and lipid transport. The DEGs also include alcohol dehydrogenase genes and pyruvate decarboxylase genes involved in sugar metabolism. In summary, the ae wx double mutations caused pleiotropic effects and transcriptional changes for a number of genes involved in metabolism, cellular response and organization. Therefore, a block in starch synthesis triggers transcriptional responses to favour the flux of excess carbohydrates into glycolysis, pentose phosphate pathway, and cell wall biosynthesis, but not toward the synthesis of alternative storage compounds. Keywords: transcriptional regulation; starch biosynthesis; carbohydrate metabolism

Introduction Starch is the most abundant storage carbohydrate in many seeds and storage organs, and exists in two different forms: the linear Į-polyglucan amylose in which the glucose units are joined end-to-end by Į-1,4 linkages and the branched Į-polyglucan amylopectin in which about 5% of the glucose units are joined by Į-1,6 linkages. The starch granule has a complex structure with a hierarchical order composed of amylose and amylopectin, which constitutes about 75% of the granule mass and allows the glucose chains to form double helices to finally pack together into * Corresponding author. Tel: +86-538-8249418, Fax: +86-538-8226399. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60092-8

semi-crystalline starch granules (Ball et al., 1996; Myers et al., 2000; Tetlow et al., 2004a). The importance of starch synthesis to plant, mankind and agriculture is never overstated. In higher plants, starch biosynthesis involves a complex dynamic process by synergies of sucrose synthase (SUS), ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE), starch debranching enzyme (DBE), and some modifying factors (James et al., 2003; Tetlow et al., 2004a; Hannah and James, 2008). In the cytoplasm of cereal endosperm, ADP-glucose is synthesized from sucrose transported from photosynthetic tissues through the catalysis by SUS, UDP-glucose pyrophosphorylase (UGPase) and AGPase sequentially. ADP-glucose is then transported into the

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amyloplast of endosperm cells through ADP-glucose translocator (BT1) in the amyloplast membrane (Shannon et al., 1998). In amyloplast, starch is synthesized from ADP-glucose by SS, SBE and DBE. The granule-bound SS (GBSS) is the only one found exclusively within the granule and is responsible for the synthesis of amylose. All three genes play distinct roles and are jointly responsible for the synthesis of amylopectin, including GBSS (Maddelein et al., 1994; van de Wal et al., 1998; Ral et al., 2006). In addition, starch synthesis is under a fine control of allosteric modulation by metabolites such as 3-phosphoglycerate (3-PGA) and inorganic orthophosphate (Pi) and of post-translational modification of the starch metabolic enzymes such as redox modification, protein phosphorylation, the formation of multiprotein complexes, and the involvement of 14-3-3 proteins (Tetlow et al., 2004b; Kolbe et al., 2005; Hennen-Bierwagen et al., 2009). In maize endosperm, mutations in a number of starch biosynthetic genes alter starch synthesis, although their effects vary depending on their genetic background. The endosperm starch lacking Wx function is composed exclusively of amylopectin (Nelson and Rines, 1962). The shrunken1 (sh1) mutation with a lesion in sucrose synthase affects the early steps in the conversion of sucrose to starch, and results in the arrest of starch synthesis and the accumulation of sugars (Koch, 2004). shtunken2 (Sh2), brittle2 (Bt2) and sugary1 (Su1) encode the large and small subunits of AGPase and isoamylase-type DBE, respectively, and their loss-of-function mutants also accumulate high levels of sugars, which inhibits the expression of pullulanase-type starch debranching enzyme (Pul) (Hannah and Nelson, 1976; Bae et al., 1990; Bhave et al., 1990; James et al., 2003). Dysfunctions of SBEIIb (amylase-extender, Ae), SSI, SSII (Dull1, Du1) and SSIIa (Sugary2, Su2) also alter starch structure to some extent (Gao et al., 1998; Yao et al., 2004; Zhang et al., 2004; Fujita et al., 2006). There has been a great accumulation of knowledge on the roles and the transcriptional regulations as well as zymological regulations in starch biosynthesis. However, relatively less is known about the transcriptional regulation for starch biosynthesis, carbohydrate metabolism and other responses due to dysfunction of genes involved in starch biosynthesis, in addition to the bt2-H2328 mutant in which the genes involved in amino acid metabolism and polysaccharide are significantly affected (Cossegal et al., 2008).

SBEIIb (ae) and GBSS (wx) encode SBE and GBSS, respectively, two key enzymes at the downstream of starch biosynthesis in the endosperm (Nelson and Rines, 1962; Cobb and Hannah, 1988; Kim et al., 1998). The ae wx mutant accumulates excessive amount of sugar at the expense of starch synthesis and has a significantly increased ratio of amylopectin to amylose (Creech, 1965). However, rice ae wx double-mutant that lack amylose have been shown to produce greatly elongated amylopectin chains that have mistakenly considered as amylose (Nishi et al., 2001). In this study, we used the endosperm of the ae wx double-mutant at 15 days after pollination (DAP) and analyzed the transcriptional changes by the dysfunctions in SBEIIb and GBSSI. We have identified more than one thousand differentially expressed genes (DEGs) as a consequence of the two mutations. The Gene Ontology (GO) terms of nucleosome assembly, lipid transport, alcohol dehydrogenase and pyruvate decarboxylase were enriched. The starch biosynthesis, carbohydrate metabolism and amino acid metabolism were also altered significantly. Our data should provide strong evidence that restrained starch biosynthesis causes a subsequent accumulation of various sugars.

Materials and methods Microarray platform The 18K Genechip® Maize Genome Array was designed by using EST contig sequences derived from multiple inbred lines. In most cases, fifteen oligonucleotide probes were a contiguous adjacent sequence of a designated gene. This array system contains 17,622 probe sets and was designed to detect the expression of 13,495 genes, in which some genes were represented by multiple probe sets to detect sense and antisense expression or the expression of alternative transcripts (Stupar et al., 2007).

Plant growth and tissue collection The maize inbred lines B73 and ae wx (with nuclear background of Hi27) were planted in the field of the experimental station in Shandong Agricultural University in the summer of 2007. In late August, the endosperm in the middle of self-pollinated ears of B73 and ae wx was collected at about 10 a.m. on 15 DAP when starch was being

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rapidly synthesized. Three different ears of each inbred line, which were similar to each other in size and shape, were used for microarray and qRT-PCR analysis as three biological replicates. The endosperm was collected and frozen in liquid nitrogen and stored subsequently at –70°C.

Endosperm starch and sugar content About 0.1–0.2 g of frozen endosperm tissues at 15 DAP of B73 and ae wx double-mutant was ground for starch extraction. Starch contents of the endosperm were measured using a amylose/amylopectin kit according to the manufacturer’s instructions (Megazyme, Ireland). About 0.6 g of tissues was ground to prepare plant powders. Five milliliters of sterilized water was then added to extract sugar from the plant powders and incubated for 12 h. Moderately-activated carbon was added to each extract after centrifugation for 15 min. After a further centrifugation for 5 min, each preparation was purified three times through a C18 solid extraction column (3 mL/50 mg). Sugar content was determined by HPLC column (4.6 mm × 250 mm, 4 m, Waters Incorporation) with 75% acetonitrile as the mobile phase. Mean linear velocity of the mobile phase was 1.0 mL/min. Rhamnose, fructose, mannitol, glucose, sucrose and a blank were measured to make standard curves. Various sugar contents were determined by CFS = 0.2 × S/SS × V × 1 g/L/m, in which CFS represents sugar content of a fresh sample, S represents the area of a sample apex, SS represents the area of a standard sample apex, V represents the volume of an extraction, and m represents the mass of a sample.

RNA isolation and microarray hybridization Total RNA was isolated by using a RNA Extraction Kit purchased from Beijing Autolab Biotechnology Co Ltd, and was cleaned up by using a RNeasy MinElute Cleanup Kit (Qiagen, USA). All RNA samples were quantified by using a Nanodrop spectrophotometer (Nanodrop Technologies, USA) and the quality of the RNA preparation was examined by agarose gel electrophoresis. The target label and microarray hybridization were performed by using Affymetrix® Hybridization Oven 640, Affymetrix® Fluidics Station 450, MessageAmpTM II-Biotin, a Poly-A RNA Control Kit, and a Hybridization Control Kit according to the Affymetrix Genechip Expression Analysis-Technical Manual. Hybridizations were performed for

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three biological replicates from three different ears of each inbred line.

Microarray data acquisition and statistical analysis An Affymetrix GeneChip Scanner 3000 was used to scan signals from the microarray images. GeneChip Operating Software Version 1.4 (GCOS 1.4) was used to produce a Molecule Annotation System 4.0 (MAS 4.0) signal and presence-absence calls. Normalization was performed separately for each chip to avoid the introduction of dependencies among biological replications by using dChip 2006 software. The data were analysed using MAS 4.0. The Cluster 3.0 program was used to perform a hierarchical clustering analysis using average linkage to create gene trees on the basis of specified gene lists, normalized MAS4.0 signal, and presence-absence calls after filtration. The trees were displayed by TreeView program (Eisen et al., 1998). DEGs were identified using Significance Analysis of Microarray (SAM) 2.10 using parameters of two-class unpaired t-tests, false difference rate (FDR) İ 0.05 and fold change (FC) ı 2.0. GO analysis was carried out by using the online Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) (Zheng and Wang, 2008).

Quantitative real time RT-PCR (qRT-PCR) The DEGs were confirmed through qRT-PCR analysis. The corresponding primers were designed using Beacon Designer 7 software. The optimal primer was designed by selecting SYBR Green Design, BLAST sequence search, template structure search, and then primer search. Only the primers that give rise to a single product and amplification efficiency up to 95%–105% were used in the validation experiment. Reverse transcription was carried out by using 2 Pg total RNA, which was from the same source as the RNA for microarray analysis. The qRT-PCR was conducted by using IQ5 quantitative PCR system (Bio-Rad). The amplicon of 18S rRNA was used as an internal control. Statistical analysis was performed by using the 2–ǻǻCT method (Livak and Schmittgen, 2001).

Annotation and classification of DEGs As the sequence length corresponding to some probe sets was not sufficiently long to get satisfactory result through BLAST search in NCBI, the TIGR Plant Tran-

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script Assemblies database was used to identify the assembled DNA sequence that contain the Representative Public ID of a probe. Only the genes that had E-value < 1e–12 in BLAST search results were considered for functional annotation. After integration of the information from different databases (i.e., NCBI, TAIR, TIGR, GO, KEGG and UniPro), each gene was designated to only one particular function such as carbohydrate/energy metabolism, amino acid metabolism or cellular organization.

Results and discussion Starch and sugar contents in endosperm The maize ae wx double-mutant (M541P) was obtained from MaizeGDB Stocks (http://www.maizegdb.org/stork. php). The mutant and the control of B73 had a similar life-cycle of around 110 days at the experimental station of Shandong Agricultural University. Since both genes play important roles in starch biosynthesis, starch accumulation was determined in the endosperm. Starch content in the fresh endosperm of the ae wx double-mutant at 15 and 25 DAP declined significantly to around 10% of those in the fresh endosperm of B73, indicating that starch synthesis was largely attenuated in the ae wx endosperm (Fig. 1A). We then investigated the defect of the ae wx double-mutant in sugar profiles of fresh endosperm at 15 and 25 DAP by using high performance liquid chromatography (HPLC). The major sugar components (including fructose, glucose and sucrose) were examined in the fresh endosperm of B73 or ae wx at 15 and 25 DAP (Fig. 1B). Much more sugars were accumulated in the fresh endosperm of ae wx than those of B73 (Fig. 1C). Sucrose constituted for approximately 60% of the major soluble sugars in the endosperm, and accumulated more than 4–5 times in ae wx than that in B73. Sucrose contents were decreased by 25 DAP compared to those at 15 DAP, suggesting that excessive carbohydrates could be redirected through alternative metabolic pathways. The glucose and fructose in the ae wx mutant were accumulated twice much as that in B73. It should be noted that the starch and sugar contents were lower than those in dry kernels in a previous report (Creech, 1965), and we determined the starch and sugar contents from fresh endosperm of B73 and ae wx in the present study. Thus, our data was generally considered to be consistent with that of the previous

report (Creech, 1965).

Microarray data analysis To further determine the effect of ae and wx mutations on large-scale transcriptional profiles, we performed the 18K Genechip® Maize Genome Array, and the microarray data in this study have been submitted to GenBank (GSE18491). After the microarray data was filtered by the digital signal and present value, a total of 8,991 probe sets were hierarchically clustered using the average linkage method of the Cluster 3.0 program. Fairly similar results were observed for the three biological replicates of both the wild type and mutant lines (Fig. 2A), and the transcriptional level of GBSSI was significantly decreased in ae wx compared to B73. But significant transcriptional alternation of SBEIIb was not detected as previous report (Stinard et al., 1993). It is possible that the lesion in ae locus is tiny (e.g., a point mutation or a frameshift) and does not interfere with transcription but either blocks translation or results in the production of a defective SBEIIb. Despite the high level of polymorphisms and structural differences among the different maize inbred lines, their transcriptomes displayed remarkable similarities especially for both samples at the reproductive stages (Ma et al., 2006). Moreover, the previous microarray data of B73 and Mo17 endosperms at 13 and 19 DAP was analyzed (Stupar et al., 2007), and there were few DEGs involved in carbohydrate and amino acid metabolism, especially in starch metabolism (Supplemental Table 1), implying a probable tiny effect of different genotypes on transcriptional regulation of genes associated with carbohydrate and amino acid metabolism. Thus, B73 was used as the control for ae wx double-mutant in this study. The DEGs were identified in ae wx compared to B73 by the two-class unpaired method of the SAM 2.10 program. By using Fold change (FC) ı 2 or İ 0.5 at FDR < 0.001 and q-value < 0.01 cutoff, a total of 1,777 probe sets were identified to account for 1,726 DEGs (Supplemental Table 2). Clustering analysis of DEGs was then performed (Fig. 2B). qRT-PCR was then used to confirm the expression changes of the DEGs (Supplemental Table 3). We further chose 30 genes for such analysis, which fell into several categories, including carbohydrate metabolism. Of them, twenty-nine genes showed similar changes to that observed in our microarray analysis although there was some minor variation in the magnitude of transcriptional change (Fig. 3).

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Fig. 1. Starch and sugar contents in fresh endosperm. A: starch contents in fresh endosperm of maize inbred line B73 and ae wx double-mutant at 15 and 25 DAP. B: biological determination of sugar HPLC profiles in B73 endosperm at 25 DAP. Fru, fructose; Glu, glucose; Suc, sucrose. C: components and contents of various sugars in fresh endosperm of maize inbred line B73 and the ae wx mutant at 15 and 25 DAP.

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Fig. 2. Cluster analysis of microarray data. The microarray data were log2-transformed and subjected to average hierarchical clustering; and up-regulated and down-regulated genes are shown in red and green, respectively. The higher the absolute value of changes, the brighter the color. The color scale is shown at bottom; grey represents the missing values. A: cluster analysis of signals from 8,991 probe sets from endosperm of two different inbred lines and three biological replicates per inbred line. B: hierarchical clustering analysis of DEGs in the ae wx mutant. DEGs are defined as significantly differential expression at least two-fold. The original data were used to categorize the 1,726 DEGs.

Gene Ontology enrichment analysis of the DEGs GOEAST program (Zheng and Wang, 2008) was used to further analyze the DEGs in the ae wx mutant (Supplemental Table 3). The genes involved in chromatin organization [GO:0006325] (P-value = 1.06E–07) were most significantly enriched in comparison to those in B73 (Table 1). For example, 27 DEGs were shown to encode histones. Among them, 25 DEGs were up-regulated and encode histone 2, 3 and 4 (Supplemental Table 4). One of two down-regulated DEG encodes histone H1 (FC, 0.01) (Supplemental Table 4). Histone H1 and histone deacetylase are the suppressors of transcription (Hecht et al., 1995; Lusser et al., 1997; Wolffe, 1997). The decrease of histone H1 may loosen the chromatin structure in ae wx mutant, which may be correlated to the corresponding transcriptional increase in the endosperm due to epigenetic regulation.

Eight DEGs that encode alcohol dehydrogenase [GO:0004022] (P-value, 0.002706) and pyruvate decarboxylase [GO:0004737] (P-value, 0.002706) were listed through the GO enrichment analysis list (Table 1). Pyruvate decarboxylase acts during fermentation to produce ethanol by converting pyruvate to acetaldehyde and carbon dioxide, and alcohol dehydrogenases facilitate the conversion of toxic alcohols to aldehydes, suggesting that the transcriptional adjustments involved in carbohydrate metabolism may be caused by the accumulation of sugar in the double mutant (Fig. 1C). In addition, twelve DEGs in lipid transport were also enriched [GO:0006869] (P-value, 0.009982) (Table 1). The transcription of four DEGs that encode physical impedance-induced proteins and are involved in lipid transport was significantly up-regulated (Supplemental Table 4). In contrast, the expression of six DEGs that encode phospholipid transfer protein or nonspecific lipid-transfer protein was considerably down-regulated.

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Annotation and classification of the DEGs To further analyze the DEGs, we annotated the differentially expressed probe sets according to their public ID for 1,162 out of the 1,726 DEGs (Supplemental Table 5). On the basis of their putative functions, the DEGs were classified into 14 groups plus an unclassified group. Among the groups, the expression of the genes involved in carbohydrate/energy metabolism, cell organization, transcription/post-transcriptional processing, stress/defence/senescence as well as protein destination/chaperone were significantly affected, accounting for 53% of the classified genes in the ae wx double-mutant endosperm (Fig. 4). The DEGs in-

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volved in starch biosynthesis, other carbohydrate and amino acid metabolism were further analyzed (Supplemental Table 6).

Starch biosynthesis There were excessive sugars at the expense of starch synthesis due to the mutations in both SBEIIb and GBSSI (Fig. 1). The expressional changes of a few genes involved in starch biosynthesis in ae wx compared to B73 were shown in Table 2. The genes encoding SBEIIa, plastid ADP-glucose pyrophosphorylase large subunit (pSH2), UGPase, G-6P translocator (GPT) and phosphoglucomutase/ phosphomannomutase (PPG/PPM) were all remarkably

Fig. 3. Confirmation of the microarray data by qRT-PCR. Thirty DEGs were selected for futher evaluation by qRT-PCR. M, Microarray data. q, qRT-PCR or semi-quantitative RT-PCR data. *, semi-quantitative RT-PCR data. #, false positive DEG by q.

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Table 1 GO enrichment analysis of differentially expressed genes GO ID

Ontology

GO Term

P-value

GO:0006325

Biological_process

Chromatin organization

1.06E-07

GO:0051276

Biological_process

Chromosome organization

1.82E-07

GO:0006323

Biological_process

DNA packaging

4.83E-07

GO:0006334

Biological_process

Nucleosome assembly

4.83E-07

GO:0031497

Biological_process

Chromatin assembly

4.83E-07

GO:0034728

Biological_process

Nucleosome organization

4.83E-07

GO:0065004

Biological_process

Protein-DNA complex assembly

4.83E-07

GO:0071103

Biological_process

DNA conformation change

7.45E-07

GO:0006333

Biological_process

Chromatin assembly or disassembly

8.16E-07

GO:0034622

Biological_process

Cellular macromolecular complex assembly

0.000125

GO:0006996

Biological_process

Organelle organization

0.000336

GO:0034621

Biological_process

Cellular macromolecular complex subunit organization

0.000388

GO:0065003

Biological_process

Macromolecular complex assembly

0.001619

GO:0022607

Biological_process

Cellular component assembly

0.001792

GO:0010926

Biological_process

Anatomical structure formation

0.003408

GO:0043933

Biological_process

Macromolecular complex subunit organization

0.003746

GO:0006869

Biological_process

Lipid transport

0.009982

GO:0010876

Biological_process

Lipid localization

0.009982

GO:0044085

Biological_process

Cellular component biogenesis

0.018244

GO:0007010

Biological_process

Cytoskeleton organization

0.081471

GO:0016043

Biological_process

Cellular component organization

0.086338

GO:0000786

Cellular_component

Nucleosome

1.35E-07

GO:0032993

Cellular_component

Protein-DNA complex

1.35E-07

GO:0000785

Cellular_component

Chromatin

1.98E-07

GO:0044427

Cellular_component

Chromosomal part

1.51E-06

GO:0005694

Cellular_component

Chromosome

4.02E-06

GO:0015629

Cellular_component

Actin cytoskeleton

0.009982

GO:0004022

Molecular_function

Alcohol dehydrogenase

0.002706

GO:0004737

Molecular_function

Pyruvate decarboxylase

0.002706

GO:0008092

Molecular_function

Cytoskeletal protein binding

0.051631

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Fig. 4. Classification of DEGs according to their biological functions. As the sequence length corresponding to some probe sets was not sufficiently long for a satisfactory result through BLAST search in NCBI, the TIGR Plant Transcript Assemblies database was used to identify assembled DNA sequence that contains the Representative Public ID of a probe. Only genes that had E-value < 1e–12 in BLAST search results were considered for functional annotation. After integration of the information from different database (NCBI, TAIR, TIGR, GO, KEGG and UniPro), each gene was designated to only one functional class according to a previous report (Li et al., 2007). The numeric values represent the percentage of the corresponding class.

up-regulated at the transcriptional level (Fig. 5, 3–6). According to our qRT-PCR data, SBEIIa was expressed predominantly in wild type maize leaves (data not shown). Loss of SBEIIa function in maize gives rise to leaf starch with little to no branching, while endosperm starch is virtually unaltered (Blauth et al., 2001; Yao et al., 2004). Surprisingly, removal of SBEIIb and SBEI from the maize endosperm increased rather than decreased the number of starch branches and decreased rather than increased starch chain length, presumably indicating that the presence of SBEI activity somehow inhibited SBEIIa activity in maize endosperm (Yao et al., 2004). However, our data in the present study showed that the transcription of SBEIIa was up-regulated in the ae wx endosperm at 15 DAP, but no transcriptional down-regulation of SBEI was observed (FC, 1.74), suggesting that SBEI may not inhibit SBEIIa at least at the transcriptional level. The up-regulation of pSH2, PPG/PPM and GPT may be caused by the accumulation of sugars in the cytoplasm of endosperm (Fig. 5). In contrast, the genes encoding for pullulanase-type starch debranching

enzyme (PUL), ȕ-amylase and sucrose synthase 3 were down-regulated (Fig. 5, 2, 7 and 8). The down-regulation of PUL expression was confirmed by the accumulation of sugars (Bhave et al., 1990; James et al., 1995). The trehalose 6-phosphate synthase gene (TPS), a positive regulatory factor gene of AGPase, was also down-regulated (Fig. 5, 9), suggesting that the allosteric properties of AGPase may function to reduce its activity in starch biosynthesis (Kolbe et al., 2005; Paul, 2007). TPS is also a key intermediate in sugar-signal transduction, which may lead to the transcriptional suppression of many genes by the presence of high level of sugars (Paul, 2007). More recent studies have demonstrated interactions for SSI, SSIIa, SSIII, SBEI, SBEIIa and SBEIIb (Grimaud et al., 2008; Hannah and James, 2008, 2009). However, the transcription of SBEIIa was not suppressed but up-regulated in the ae wx mutants (Table 2), suggesting that the mutations in SBEIIb and GBSSI did not significantly inhibit the transcription of SBEIIa, SSI, SSIIa, SSIII and SBEI. The induction of SBEIIa, normally operating in the leaf, could

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Table 2 Differentially expressed genes coding for enzymes in selected carbohydrate and amino acid metabolism pathways No. in Fig.5

EC No.

Enzyme name

Up in ae wx

Down in ae wx

1

EC 3.2.1.26

Cell wall invertase 4

1

2

EC 2.4.1.13

Sucrose synthase

3 4 5 6 7

EC 2.7.7.9 EC 5.4.2.2/8 EC 2.7.7.27 EC 2.4.1.18 EC 3.2.1.41

UDP-glucose pyrophosphorylase Phosphoglucomutase/phosphomannomutase ADP-glucose pyrophosphorylase Starch branching enzyme IIa Pullulanase-type starch debranching enzyme

8

EC 3.2.1.2

ȕ-amylase

1

9

EC 2.4.1.15

Trehalose 6-phosphate synthase

1

10

EC 2.7.1.1

Hexokinase-1

1

1 1 1 1 1 1

11

EC 2.4.1.14

Sucrose phosphate synthase

12

EC 3.1.3.24

Sucrose-phosphatase

1

13

EC 2.4.1.34

Callose synthase 1

1

1

14

EC 2.4.1.12

Cellulose synthase

2

15

EC 5.1.3.6

UDP-glucuronic acid 4-epimerase

1

16

EC 3.2.1.15

Polygalacturonase

17

EC 5.1.3.2

UDP-glucose 4-epimerase

18

EC 2.4.1.46

Monogalactosyldiacylglycerol synthase

1

19 20

EC 3.2.1.23 EC 5.1.3.18

ȕ-galactosidase GDP-mannose 3,5-epimerase 1 (NAD+)

2 1

1 1 1 1

21

EC 5.4.2.1/4

Phosphoglycerate/bisphosphoglycerate mutase

22

EC 1.1.1.27

L-lactate dehydrogenase

1

23

EC 4.1.1.36

Phosphopantothenoylcysteine decarboxylase

1

24

EC 3.1.1.31

6-phosphogluconolactonase

25

EC 1.1.1.44

6-phosphogluconate dehydrogenase

26

EC 2.2.1.1

Transketolase

1

27 28 29 30

EC 1.1.1.95 EC 2.6.1.45 EC 1.5.3.1 EC 4.2.1.20

D-3-phosphoglycerate dehydrogenase Serine-glyoxylate aminotransferase Sarcosine oxidase Tryptophan synthase

3

1 1

1 1 1

31

EC 2.3.3.13

2-isopropylmalate synthase

1

32

EC 2.6.1.42

Branched-chain-amino-acid aminotransferase

1 1

33

EC 1.2.1.25

2-oxoisovalerate dehydrogenase

34

EC 6.3.5.4

Asparagine synthetase

35

EC 1.1.1.3

Aspartate kinase-homoserine dehydrogenase

36 37 38 39 40 41 42 43 44 wx A B C

EC 2.1.1.10 EC 2.5.1.6 EC 2.1.1.12 EC 4.1.1.50 EC 2.5.1.48 EC 4.1.3.27 EC 4.2.1.51 EC 4.3.1.24 EC 4.2.1.19 EC 2.4.1.21 * * *

Homocysteine S-methyltransferase S-adenosylmethionine synthetase Methionine S-methyltransferase S-adenosylmethionine decarboxylase O-succinylhomoserine sulfhydrylase Anthranilate synthase Prephenate dehydratase Phenylalanine ammonia-lyase Imidazoleglycerol-phosphate dehydratase Granule-bound starch synthase 1 G-6P translocator Hydroxyproline-rich glycoprotein Cell wall glycoprotein

1 2 1

D

*

Cell wall protein

1

*, No EC number.

1 1 1 1 1 3 1 1 1 1 1 1

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Fig. 5. Summary of transcriptional changes in ae wx endosperm. The scheme shows selected pathways of the carbohydrate and amino acid metabolism. Starch biosynthesis, glycolysis, pentose-phosphate pathway (PPP) and cell wall metabolism are boarded by circles; the different amino acid metabolic pathways are not. Solid arrows indicate a single enzymatic step, and dashed arrows indicate more than one enzymatic step. Enzymes are numbered according to Table 2. Enzymes encoded by genes over- or under-expressed in ae wx endosperm and the putative metabolites accumulated at high or low level are highlighted in red and green, respectively. Yellow highlighting indicates that at least one corresponding gene is over-expressed and at least one corresponding gene is under-expressed. Gal, galactose; T-6P, trehalose 6-phosphate.

be due to a lack of intermediates in starch biosynthesis. These results indicate that the regulation of genes involved in starch biosynthesis pathway is rather complex.

Other carbohydrate metabolisms We next addressed the influence of the double mutations on the expression of genes involved in other carbohydrate metabolisms. We found more DEGs involved in other carbohydrate metabolic processes than those involved in starch synthesis (Table 2). The down-regulated genes encode sucrose synthase and hexokinase (Fig. 5, 2

and 10) and one up-regulated gene encodes invertase (Fig. 5, 1), consistent to the accumulation of sucrose, glucose and fructose (Fig. 1). Among the six characterized maize invertase genes (Kim et al., 2000), only cell wall invertase 4 (IncW4) was up-regulated (Table 2). IncW4 is constitutively expressed and may be a new type of cell-wall invertase present in a free form in the apoplast. Since IncW4 is a cell wall invertase, its overexpression leads to increased, irreversible sucrose cleavage in the apoplast and consequently to increased extracellular levels of glucose and fructose (Cossegal et al., 2008).

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A more precise dissection of the metabolic pathways that are turned on or off in the mutant endosperm is hampered because of the complexity of metabolic network and the limitation of public data. Nevertheless, some general trends of metabolism emerged (Fig. 5, 1320). The up-regulation of one DEG coding for phosphomannomutase and 2 DEGs coding for GDP-mannose 3,5-epimerase 1 (NAD+) increased the synthesis of GDP-L-galactose (Goudsmit and Neufeld, 1967). The callose and cellulose biosynthesis might increase due to the up-regulation of the genes coding for callose synthase 1 (Schlupmann et al., 1994) and cellulose synthase (Somerville, 2006). One up-regulated gene encodes UDP-glucuronic acid 4-epimerase and may lead to increased pectin biosynthesis, whereas the gene encoding polygalacturonase and functioning in the pectin catabolism was down-regulated (Zheng et al., 1994). Due to an up-regulation on gene encoding monogalactosyldiacylglycerol synthase, the galactosyl diacylglycerol should be accumulated, together with the down-regulation of genes encoding ȕ-galactosidase (Dhugga, 2005). Furthermore, we found four up-regulated genes encoding cell wall proteins (Fig. 5, B, C and D), and cell wall biosynthesis may be one of the flux of excess sugars (Fig. 5). In addition, there were seven up-regulated genes and five down-regulated genes involved in glycolysis and the pentose phosphate pathway (PPP) (Table 2 and Fig. 5). It is rather difficult to discuss all the up- and down-regulated genes individually because our knowledge is still limited on the gene families encoding enzymes involved in glycolysis and PPP. However, the transcription of one gene encoding phosphoglycerate/bisphosphoglycerate mutase was affected in association with a possible increase in glycolysis and the enhancement of pyruvate catabolism (Fig. 5). Likewise, PPP was possibly intensified due to 2 up-regulated genes that encode 6-phosphogluconate dehydrogenase and transketolase, respectively (Fig. 5).

Amino acid metabolism Amino acid metabolism was also significantly affected in the ae wx double-mutant (Fig. 5). Because of the complexity of amino acid metabolism and its inter-connections with glycolysis, TCA and PPP, it is also difficult to discuss all amino acid metabolism individually (Fig. 5). Nevertheless, the transcriptional changes occurred for a few genes involved in amino acid metabolism in our current studies.

The up-regulation of three DEGs that encode D-3-phosphoglycerate dehydrogenase has been shown to result in increased serine biosynthesis (Yamasaki et al., 2001). We found three downregulated genes involved in serine catabolism (Fig. 5, 28–30). These changes together may lead to an increased synthesis of serine (Fig. 5, 27–30) and a decreased synthesis of glycine (Fig. 5, 28–29) and tryptophan (Fig. 5, 30 and 41). We also noticed that the genes involved in the biosynthesis of methionine and phenylalanine were down-regulated (Fig. 5, 36 and 42), and the genes involved in their degradation were up-regulated (Fig. 5, 37, 38 and 43). As a result, the accumulation of these compounds may decrease significantly (Azevedo et al., 2006; Less and Galili, 2008). In addition, the genes related to the biosynthesis and catabolism of valine, leucine and isoleucine were down-regulated together (Azevedo et al., 2006; Stepansky and Leustek, 2006). In our studies, eight DEGs encoding amino acid transporters were up-regulated in the ae wx mutant (Supplemental Table 5). We suggest that the differential regulation of amino acid metabolism may be due to their different functions in cellular metabolism rather than general protein biosynthesis. In addition to metabolism, a number of DEGs detected in the ae wx mutant were found to be involved in cell responses (Fig. 4). We found that 35 DEGs encode ribosomal protein or RNA, and 30 of them were significantly up-regulated (Supplemental Table 7). Ribosomal proteins are ubiquitous, abundant and RNA-binding, most likely as priming candidates for the recruitment to extra-ribosomal functions other than protein synthesis in spite of very few verified cases in the self-evaluation of cellular health and cell-cycle arrest or apoptosis (Warner and McIntosh, 2009). The regulation of 21 genes involved in cell cycle control were also affected in the double mutant (Supplemental Table 7). Taken together, the microarray data strongly suggest that a block in starch biosynthesis due to the mutations in SBEIIb and GBSSI triggers the transcriptional regulation of many genes to alter the flux of excess carbohydrates into various pathways, such as glycolysis, pentose phosphate pathway, and cell wall biosynthesis. We did not observe a concerted shift to the synthesis of alternative storage substances. Furthermore, a combination of the genome-wide protein profile analysis with enzymatic assays might help to reveal the starch-mediated regulation on many biological processes and metabolism pathways.

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Acknowledgements This work was supported by grants from the Ministry of Agriculture of China (Nos. 2008ZX08003-003 and 2009ZX08003-023B) and Shandong Agricultural University (No. 23651). We are grateful to the Maize Genetics Cooperation Stock Center for the seeds of the ae wx double-mutant.

Supplemental data Supplemental Tables 17 associated with this article can be found in the online version at www.jgenetgenomics.org.

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