Ultrastructural observation of mesophyll cells and temporal expression ...

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Using transmission electron microscopy, we observed that during the day after anthesis, starch granules in mesophyll cells of wheat flag leaves accumulated in ...
Physiologia Plantarum 153: 12–29. 2015

© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Ultrastructural observation of mesophyll cells and temporal expression profiles of the genes involved in transitory starch metabolism in flag leaves of wheat after anthesis Guozhang Kanga* , Xiaoqi Penga , Lina Wangb , Yingying Yangb , Ruixin Shaoa , Yingxin Xiec , Dongyun Mab , Chenyang Wangc , Tiancai Guoa,c and Yunji Zhua,* a

The Collaborative Innovation Center of Henan Food Crops, Henan Agricultural University, Zhengzhou 450002, China The National Engineering Research Centre for Wheat, Henan Agricultural University, Zhengzhou 450002, China c The National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450002, China b

Correspondence *Corresponding authors, e-mail: [email protected]; [email protected] Received 4 April 2014 doi:10.1111/ppl.12233

Transitory starch in cereal plant leaves is synthesized during the day and remobilized at night to provide a carbon source for growth and grain filling, but its mechanistic basis is still poorly understood. The objective of this study is to explore the regulatory mechanism for starch biosynthesis and degradation in plant source organs. Using transmission electron microscopy, we observed that during the day after anthesis, starch granules in mesophyll cells of wheat flag leaves accumulated in chloroplasts and the number of starch granules gradually decreased with wheat leaf growth. During the night, starch granules synthesized in chloroplasts during the day were completely or partially degraded. The transcript levels of 26 starch synthesis-related genes and 16 starch breakdown-related genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. Expression profile analysis revealed that starch metabolism genes were clustered into two groups based on their temporal expression patterns. The genes in the first group were highly expressed and presumed to play crucial roles in starch metabolism. The genes in the other group were not highly expressed in flag leaves and may have minor functions in starch metabolism in leaf tissue. The functions of most of these genes in leaves were further discussed. The starch metabolism-related genes that are predominantly expressed in wheat flag leaves differ from those expressed in wheat grain, indicating that two different pathways for starch metabolism operate in these tissues. This provides specific information on the molecular mechanisms of transitory starch metabolism in higher plants.

Introduction Starch is one of the major storage carbohydrate reserves in higher plants (Zeeman et al. 2010). The two types of starch are storage starch and transitory starch. In

heterotrophic storage organs, such as cereal grains or potato tubers, starch is remobilized later in development to support germination (Geigenberger 2011). In photosynthesizing organs, including green leaves, transitory starch accumulates during the day and is remobilized

Abbreviations – AGPase, ADP-Glc pyrophosphorylase; AMY, 𝛼-amylase; BAM, 𝛽-amylase; BE, starch branching enzyme; DBE, starch debranching enzyme; DPE, disproportionating enzyme; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBSS, granule-bound starch synthase; Glc, glucose; GWD, glucan water dikinase; LSU, large subunits; MDA, malondialdehyde; PHO, phosphorylase; PPB, potassium phosphate buffer; PUL, limit dextrinase; PWD, phosphoglucan water dikinase; qPCR, quantitative real-time polymerase chain reaction; SS, soluble starch synthase; SSU, small subunits.

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at night to support continued respiration, Suc export and growth in the dark (Lu et al. 2005, Geigenberger 2011). Starch consists of two distinct types of glucose (Glc) homopolymers: amylose and amylopectin. Amylose is essentially a linear molecule in which glucosyl monomers are joined via 𝛼-1,4 linkages. Amylopectin, the more abundant polymer in starch, has a much more defined structure (tandem cluster) than glycogen; it is composed of linked tandem clusters (approximately 9–10 nm each in length) in which linear 𝛼-1,4-glucan chains are regularly branched via 𝛼-1,6-glucosidic linkages (Dian et al. 2005). Many enzymes involved in starch metabolism (biosynthesis and degradation) have already been investigated. Amylose is synthesized by ADP-Glc pyrophosphorylase (AGPase) and granule-bound starch synthase (GBSS), while amylopectin is synthesized by the coordinated actions of AGPase, soluble starch synthase (SS), starch branching enzyme (BE) and starch debranching enzyme (DBE) (Hwang et al. 2005, Jeon et al. 2010). Disproportionating enzyme (DPE) and phosphorylase (PHO) may function in starch biosynthesis, although they are generally considered to be involved in starch degradation (Ohdan et al. 2005, Jeon et al. 2010). Starch degradation is the phosphorylation of a portion of the glucosyl residues by glucan water dikinase (GWD), phosphoglucan water dikinase (PWD), 𝛼- and 𝛽-amylases (AMY and BAM), limit dextrinase (PUL) and 𝛼-glucan PHO (Radchuk et al. 2009). Transitory starch differs from storage starch as it has apparent polymodality within the short chain fraction that forms the first population of the polymodal distribution (Hizukuri 1986). This difference implies a divergence in starch synthesis and degradation (Santacruz et al. 2004). Biosynthesis and degradation have been most intensively studied in storage starch in non-photosynthetic starch-storing organs, such as developing seeds and tubers (Ohdan et al. 2005, Radchuk et al. 2009, Yan et al. 2009); however, transitory starch synthesized in photosynthetic organs (leaves) has received less attention. Transitory starch also plays many important roles in plants. First, it may act as an overflow for newly assimilated carbon when assimilation exceeds the demand for Suc. Second, it may provide a source of carbon for growth during the night (Lu et al. 2005, Weise et al. 2011). While plants can live without transitory starch, they are at a disadvantage. When transitory starch metabolism is blocked through mutagenesis, the effects on the plant depend on where the blockage occurs. Completely blocking transitory starch synthesis, for example by genetic inactivation of plastidic phosphoglucomutase or AGPase, results in severe stunting and delayed flowering (Caspar et al. 1985,

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Lin et al. 1988a, 1988b, Slewinski et al. 2008, Weise et al. 2011). Most enzymes involved in starch metabolism in higher plants have multiple isoforms (Ohdan et al. 2005). The number of isoforms for each enzyme is plant species-specific and the expression profiles of many isoforms are diverse among plant species. For example, soluble starch synthase IVb (SSIVb) exists in rice, but is not found in maize (Ohdan et al. 2005, Yan et al. 2009). AMY is highly expressed in rice leaves, demonstrating that it plays an important role in starch degradation in this plant species (Asatsuma et al. 2005), whereas it is expressed at low levels in Arabidopsis leaves (Yu et al. 2005). In addition, the roles of some isoforms are species-specific. For example, distinct functional properties for the ISA1 and 2 genes in monocot and dicot leaves have recently been identified (Facon et al. 2013). Thus, analysis of the expression patterns of all starch metabolism genes is required to clarify the features of developmental stage-specific starch biosynthesis and degradation in important food crops (Nakamura 2002, Ohdan et al. 2005, Hannah and James 2008). The number and expression profiles of the genes involved in transitory starch biosynthesis and degradation in the leaves of Arabidopsis, a model plant, have been extensively investigated (reviewed by Grennan 2006, Zeeman et al. 2007a, Stitt and Zeeman 2012). However, expression profiles of genes or enzymes for transitory starch metabolism in important crops are poorly understood. Expression profiles of metabolic pathways can be measured at transcriptional and enzyme levels. Because every starch metabolic enzyme class has multiple isoforms or subunits and these enzymes are usually labile, separately measuring the protein level or activity of each enzyme in the enzyme complex is difficult, especially in the presence of interfering enzymes such as hydrolytic enzymes. Thus, transcriptome analysis is a more convenient tool to reveal the regulatory mechanism for starch metabolism that determines when and where multiple genes are co-expressed and what proteins may interact although there may be posttranscriptional and posttranslational controls (Smith et al. 2004, Ohdan et al. 2005). The transcript profiles of the genes involved in starch synthesis and degradation have been performed in rice and barley grains (the major starch storage organ) (Ohdan et al. 2005, Radchuk et al. 2009). To our knowledge, however, no prior study has investigated the expression profiles of transitory starch biosynthesis and degradation in the photosynthetic organs of important crops. Wheat is one of the most important crops globally, occupying the largest portion of worldwide cultivated acreage (about 20% of the total arable area) and feeding about 40% of the world population by providing 20% of the 13

total food calories and protein to the human diet (Gupta et al. 2008). The flag leaf is the major organ of transitory starch metabolism in this species and its assimilates are the most important contributor to dry weight accumulation in the grains (Yang et al. 2007, Ali et al. 2010). We hypothesized that transitory starch biosynthesis and degradation in plant leaf could be regulated by many genes, and these genes could show differential expression profiles during starch metabolism. The objective of this study was to explore the regulatory mechanism for starch biosynthesis and degradation by determining transcription profiles of the gene families involved in transitory starch synthesis and degradation in the flag leaves of common wheat. These data will help us understand starch metabolism in higher plants and possibly pave the way to new strategies to improve crop yield via the use of genetic breeding.

Materials and methods Plant materials A common winter wheat cultivar (Zhoumai 18) was grown in the field of the Agricultural and Experimental Farm of Henan Agricultural University (Zhengzhou, China; 34∘ 92′ N, 112∘ 99′ E) during the wheat growing season (October 2012 to June 2013). The soil type at the study sites was a calcareous fluvo-aquic soil with a loamy and silt texture, containing 1.2% organic matter and available nitrogen–phosphorus–potassium at 902.2, 22.1 and 240.3 mg kg−1 , respectively. The plot dimension was 4 × 8 m and three plots were separated by a ridge (20 cm in width). Seeds were sown on October 15, 2012 and the plant density was adjusted to 150 plants m−2 at the three-leaf stage. Phosphate (75 kg hm−2 P2 O5 ) and potassium (60 kg hm−2 K2 O) fertilizers were applied before sowing, whereas 50% N fertilizers (90 kg hm−2 N) were applied before sowing and the remaining 50% N fertilizers were applied with irrigation at the jointing stage. Two hundred flag leaves that headed on the same day of anthesis were chosen and marked for each plot. Meteorological parameters (rainfall, temperature, etc.) after anthesis in the experimental field are indicated in Table S1. The marked flag leaves from each plot were harvested at 06:30, 11:00, 18:30 and 23:00 h at each sampling time point after anthesis at 4-day intervals. At every sampling time point, about 45 flag leaves from different plants were harvested. Fresh leaves were excised and fixed in cold 4% (v/v) glutaraldehyde in 0.1 M potassium phosphate buffer (PPB, pH 7.2) for electron microscopic observation. The remaining sampled leaves were rapidly frozen in liquid nitrogen for 2 min and then stored at 14

−80∘ C for measurement of physiological parameters and a transcription-level analysis of the genes involved in starch synthesis and degradation enzymes. Measurement of photosynthetic pigment and MDA content Photosynthetic pigment contents were determined in 80% acetone leaf extracts and calculated according to the method of Lichtenthaler (1987). Malondialdehyde (MDA) content was determined as described previously (Zheng et al. 2008). Frozen samples (0.5 g) mixed with 5 ml phosphate buffer (pH 7.8) were crushed into a fine powder in a mortar and pestle under liquid nitrogen. The homogenate was centrifuged at 10 000 g for 20 min at 4∘ C. A mixture of 1 ml extract and 2 ml of 0.6% (w/v) thiobarbituric acid was boiled at 100∘ C for 15 min, cooled and centrifuged at 10 000 g for 10 min. The absorbance of the supernatant was measured with a UNICO [UV-2600; UNICO (Shanghai) Instruments Co., Ltd., Shanghai, China] spectrophotometer. MDA content was calculated from UV absorbance at 600, 532 and 450 nm. Determination of transitory starch content, electron microscopic observation of mesophyll cells and transcription-level assay of the genes encoding starch synthesis at the daytime and degradation enzymes at following nights in wheat flag leaves Transitory starch metabolism in plant leaves is highly regulated, and favored at high rates of photosynthesis, with synthesis increasing with a rising photosynthetic rate (Weise et al. 2011). Under field conditions in north China, the day and night cycles are usually 06:30–18:30 and 18:30–06:30 h, respectively, after wheat plant anthesis. In this region, the photosynthetic rate in most wheat cultivars peaks at about 11:00 h each day after anthesis (Ma et al. 2013), implying that the highest rate of transitory starch synthesis in flag leaves also occurs at this time. Therefore, in this study, transcription levels of the genes encoding starch synthesis were measured at 11:00 h (4 1∕2 h after daybreak) for each sampling time point, when the highest rates of both leaf photosynthesis and transitory starch synthesis occur. Transitory starch is gradually synthesized and accumulated in leaves during the day and starch levels peak at the end of the day (Streb and Zeeman 2012). Accordingly, the leaf transitory starch content was measured and leaf mesophyll cell ultrastructure was observed at the end of the day (approximately 18:30 h) for each sampling time point. In a previous study, to identify genes encoding starch synthesis enzymes in wheat, we performed sequence

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similarity searches with publicly available sequences from monocot plants using BLASTN, and the ESTs with high percentages of identity were selected. To define gene family members, rice (Oryza sativa) and maize (Zea mays) genome sequence annotations were used. Thus, 26 putative cDNA sequences of genes involved in starch synthesis were found in common wheat (Table S2). Quantitative real-time polymerase chain reaction (qPCR) was used to measure the transcription levels of the genes encoding starch synthesis enzymes in wheat flag leaves. Total RNA was also extracted from the frozen flag leaves at 0, 5, 10, 15, 20, 25, 30 and 35 days after anthesis using TRIzol® RNA Isolation Reagent (Invitrogen, Carlsbad, CA), followed by treatment with RNase-free DNase I [Takara Biotechnology (Dalian) Co., Ltd., Dalian, China] to remove contaminating genomic DNA. The integrity of the RNA samples was confirmed by gel electrophoresis. cDNA was synthesized from 2 μg of total RNA using ThermoScript Reverse Transcriptase (Invitrogen) and an oligo (dT18 ) primer at 42∘ C according to the manufacturer’s protocol. qPCR was performed using a SYBR Premix Ex Taq (Perfect Real Time) Kit [Takara Biotechnology (Dalian) Co., Ltd.] in a LightCycler® 480 Real-Time PCR System (Roche Diagnostics Ltd., West Sussex, UK) according to the manufacturer’s instructions. Each reaction (20 μl) consisted of 10 μl SYBR Green Supermix (2×), 1 μl diluted cDNA and 0.5 μl forward and reserve primers. The conserved sequences for starch synthesis genes were used to design primers for detecting gene transcript levels in wheat flag leaves by qPCR (Table S2). To verify the specificity of each primer set, the amplification products were cloned in the pMD18-T vector [Takara Biotechnology (Dalian) Co., Ltd.] and confirmed by DNA sequencing. The relative transcript levels of the target genes were calculated using the 2−ΔΔCt method with two internal control genes, wheat 𝛽-actin (actin) (GenBank Accession No. AB181991) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (GenBank Accession No. EF592180). No significant starch degradation occurs during the light period (Smith et al. 2004), and thus, transcripts of the genes encoding transitory starch degradation enzymes were not measured in the day for each sampling time point after anthesis. Leaf transitory starch content was determined following the method of Zhao et al. (2008). Flag leaves (200–400 mg dry weight) were boiled in 50 ml 80% (v/v) ethanol. The decolored leaves were then ground with a mortar and pestle in 80% ethanol and centrifuged for 10 min at 12 000 g; the pellet was washed twice with 80% ethanol. After centrifugation, the insoluble material was suspended in 1 ml of distilled water and boiled

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for 30 min. Total starch in each leaf sample was quantified using a commercial Glc assay kit (R-Biopharm, Darmstadt, Germany) according to the manufacturer’s protocol. Samples of flag leaf blades were collected randomly from four plants at 18:30 h in the afternoon (approximately at the end of the day) for electron microscopic observation. Sample sections from the middle portion of the flag leaves (1 mm2 , top middle section) were excised and fixed in cold 4% (v/v) glutaraldehyde in 0.1 M PPB (pH 7.2), vacuum-infiltrated until the material sank and left overnight at 4∘ C. Samples were then washed in five changes of 0.1 M PPB buffer (20 min each) and post-fixed in 1% osmium tetroxide [OsO4 , Osmium (VIII) oxide] for 16 h at 4∘ C in 0.1 M PPB buffer. The materials were washed with five changes of 0.1 M PPB buffer, dehydrated in a graded ethanol series (10, 20, 30, 40, 50, 60, 70, 80, 90, 95 and 100%) for 15–20-min intervals, followed by acetone (100%) for 20 min and then infiltrated and embedded in Spurr’s resin overnight. Ultrathin sections of about 80–90 nm were obtained with a diamond knife on an EM US6 ultramicrotome (Leica, Wetzlar, Germany) and then stained with 2% uranyl acetate for 90 min and 6% lead citrate for 10 min. Sample semimicrosections of 0.2 μm were examined with a transmission electron microscope (model 7500; Hitachi, Tokyo, Japan) at 80 kV. The number of starch granules was recorded in >10 mm2 of sections from each plant according to the method of Kong et al. (2010). At least five sections from each sampling time point were examined. In photosynthetic tissues, including flag leaves, transitory starch accumulated during the day is progressively degraded into Glc and maltose during the following night, both of which are exported to the cytosol for further metabolism (Stitt and Zeeman 2012). During the night, transitory starch is broken down at a relatively constant rate and is almost exhausted by the end of the night (Zeeman et al. 2007b). Thus, the transcription levels of the genes encoding transitory starch degradation were measured at 23:00 h (approximately 4 1∕2 h after nightfall) in this study. Transitory starch content and ultrastructural observations of starch granules were also measured and checked at 06:30 h the following day (approximately the end of the night) using the above methods. Barley and wheat are close relatives and most of the genes in these two species have high similarity. To identify genes encoding starch degradation enzymes in wheat, we performed sequence similarity searches using the barley genes involved in transitory starch degradation and selected related sequences in common wheat with high identity percentages (Table S3). qPCR was also used to measure the transcription levels of the genes encoding 15

ANOVA using the SPSS 17.0 statistical software (SPSS Inc., Chicago, IL) and Duncan’s multiple range test were used to identify significant (P < 0.05) differences between group means.

Results

35 days

30 days

25 days

20 days

15 days

10 days

5 days

0 days

Changes in phenotype and chlorophyll pigment and MDA contents in wheat flag leaves after anthesis

Fig. 1. Phenotypic changes of wheat flag leaf at 0, 5, 10, 15, 20, 25, 30 and 35 days after anthesis.

starch degradation enzymes in wheat flag leaves at night. The names of the genes encoding starch degradation enzymes in wheat and the primer sequence of those genes are listed in Table S3. Actin and GAPDH were used as control genes to evaluate the relative transcription levels of the target genes. Electron microscopic observations of flag leaf mesophyll cells at 06:30 h (approximately at the end of the dark period) were performed following the same procedure. Statistical analysis All measurements were repeated independently in three biological replicates with three technical replicates (three flag leaves per technical replicate). A one-way

After anthesis, when starch and other constituents begin to be synthesized and accumulate in crop grains (Jeon et al. 2010, Kang et al. 2013), wheat flag leaves gradually became yellow (Fig. 1). These phenotypic results were confirmed by quantitative analysis. Chlorophyll pigment content in wheat flag leaves decreased gradually to 20 days after anthesis and quickly declined thereafter (Fig. 2A). Moreover, MDA content quickly increased as flag leaves grew old (Fig. 2B). At 35 days after anthesis, chlorophyll pigment content in flag leaves declined by 85.3%, but MDA content increased by 82.2%. Ultrastructures of mesophyll cells in wheat flag leaves during the day and in the following night after anthesis Transmission electron micrographs of flag leaf mesophyll cells at the end of the day of each sampling time point after anthesis are shown in Fig. 3. At the end of the day (approximately 18:30 h) during the initial stages of anthesis, flag leaf mesophyll cells were characterized by well-differentiated chloroplasts containing fully developed grana having numerous layers and well-developed stroma lamellae with several starch granules and a small number of plastoglobuli (Fig. 3A, C, E, G). As wheat leaf growth progressed, the thylakoid membranes of chloroplasts began to loosen slightly, with an irregular

Fig. 2. Contents of chlorophyll (A) and MDA (B) in wheat flag leaves after anthesis. Bars represent standard errors of triplicate experiments.

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Fig. 3. Electron microscope micrographs of wheat leaf mesophyll cells of each sampling time point after anthesis with 4-day intervals. A, C, E, G, I, K, M and O, chloroplasts of mesophyll cells at the end of daytime (approximately 18:30 h) at 0, 5, 10, 15, 20, 25, 30 and 35 days after anthesis, respectively; B, D, F, H, J, L, N and P, chloroplasts of mesophyll cells at the following night (approximately 06:30 h) at 0, 5, 10, 15, 20, 25, 30 and 35 days after anthesis, respectively. CH, chloroplast; CW, cell wall; GT, grana thylakoid; M, mitochondria; N, nucleus; P, plastoglobule; S, starch; SD, starch with incomplete degradation. White lines of black panes express the bars of magnification.

arrangement of thylakoid stacks and the appearance of some large vesicles among the lamellae. In addition, the amount of starch declined, while the number of plastoglobuli markedly increased (Fig. 3I, K). In the final stages (30 and 35 days after anthesis), the chloroplasts became smaller and structurally swollen. Chloroplast envelopes disintegrated and thylakoid grana were disrupted and irregularly arranged (Fig. 3M, O). As leaf growth progressed after anthesis, the number of chloroplasts and starch granules declined gradually, whereas considerable amounts of plastoglobuli accumulated in chloroplasts (Fig. 4A–C). Compared with cell micrographs at the end of the day of each sampling time point, no notable changes, except for starch granules, were observed in the ultrastructure of mesophyll cells at the end of the following night (approximately 06:30 h) (Fig. 3B, D, F, H, J, L, N, P). At many sampling time points after anthesis, many

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incompletely degraded starch granules were clearly observed in chloroplasts at the end of the night (Fig. 3B, H, J, L). Similar to observations at the end of the day, the number of plastoglobules in chloroplasts also increased remarkably during the following night as leaf growth progressed after anthesis.

Transitory starch contents and transcription levels of genes encoding starch synthesis and degradation enzymes during the days and following nights after anthesis in flag leaves, respectively At the beginning of anthesis, transitory starch content in flag leaves was high, reaching a maximum around 5 days after anthesis, then gradually declining as leaves progressed toward senescence. Transitory starch synthesis in flag leaves after anthesis was divided into two stages: 17

Fig. 5. Contents of transitory starch in wheat flag leaves at the end of daytime and the following night at each sampling time point after anthesis. Bars represent standard errors of triplicate experiments.

the daytime, although they had similar changes during anthesis (Fig. 5). In common wheat, 26 putative cDNA sequences of starch synthesis genes were found (Table S2). The expression profiles of these genes in wheat flag leaves after anthesis at 11:00 h in the morning were analyzed by qPCR using the actin gene as an internal control (Table 1) and are described below. Similar results were obtained using GAPDH as another internal control as indicated in Table S4. ADP-Glc pyrophosphorylase

Fig. 4. Temporal changes in the number of chloroplasts per mm2 cell profile (A), granules per chloroplast (B) and plastoglobules per chloroplast (C) in flag leaf mesophyll cells at the end of daytime of each sampling time point after anthesis. The number of chloroplasts is the mean ± SD of four plants (>10 mm2 of sections were recorded from each plant). The number of starch granules per chloroplast is the mean ± SD of four plants (>20 cells with the largest dimension were recorded from each plant). The number of plastoglobule per chloroplast is the mean ± SD of four plants (>50 chloroplasts with the largest dimension were recorded from each plant).

highly accumulating and remaining at high levels about 0–20 days after anthesis, then quickly declining to low levels thereafter (Fig. 5). Transitory starch contents in flag leaves in the following night of each sampling time point after anthesis were remarkably lower than those in 18

AGPase in monocots is a heterotetramer composed of two large subunits (LSU) and two small subunits (SSU) with cytosolic and plastidial isoforms (Tetlow et al. 2004). In wheat, SSU is encoded by TaAGPS1 and TaAGPS2. TaAGPS1 encodes both the cytosolic and plastidial isoforms (TaAGPS1-a and TaAGPS1-b), which are produced by alternative splicing. The TaAGPS1-b and OsAGPS2 translation products are predicted to be plastid-targeted, while the OsAGPS1-a-encoded subunit is cytosolic. In total, three SSUs (TaAGPS1-a, TaAGPS1-b and TaAGPS2) and two LSUs (TaAGPL1 and TaAGPL2) are identified in wheat (Table S2) (Burton et al. 2002, Rösti et al. 2006, Kang et al. 2013). TaAGPL2 and TaAGPS2, which encode the LSU and SSU, respectively, of plastidial AGPase, were highly expressed at the beginning of anthesis, rose to a peak 5 days later and then declined thereafter. Transcript levels of these two genes coincided temporally with transitory starch content in flag leaves (Table 1). This indicated that both TaAGPL2 and TaAGPS2 might associate to form a heterotetrameric plastidial AGPase, which plays an

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Starch degradation-related genes

Starch synthesis-related genes

Gene names TaAGPS1-b TaAGPS2 TaAGPL1 TaAGPL2 TaGBSS I TaGBSSII TaSSI TaSSIIb TaSSIIc TaSSIIIa TaSSIIIb TaSSIV TaBEI TaBEIIa TaBEIII TaISAI TaISAII TaISAIII TaPUL TaPHOL TaPHOH TaDPE1 TaDPE2 TaAMY1 TaAMY2 TaAMY4 TaBAM2 TaBAM3 TaBAM4 TaBAM5 TaBAM6 TaPWD TaPHOL TaPHOH TaDPE1 TaDPE2

0.39 ± 0.02 7.86 ± 0.03 0.55 ± 0.09 14.70 ± 0.54 0.49 ± 0.06 3.05 ± 0.19 6.59 ± 0.37 3.30 ± 0.56 0.58 ± 0.12 0.62 ± 0.08 14.94 ± 1.34 4.41 ± 0.30 1.69 ± 0.10 3.24 ± 0.17 1.52 ± 0.13 1.00 ± 0.11 0.33 ± 0.03 0.65 ± 0.03 0.78 ± 0.05 9.80 ± 0.58 0.25 ± 0.08 0.84 ± 0.08 3.94 ± 0.27 0.18 ± 0.08 0.01 ± 0.00 8.18 ± 0.40 0.14 ± 0.01 0.83 ± 0.10 0.27 ± 0.10 3.07 ± 0.25 4.47 ± 0.36 1.64 ± 0.12 7.64 ± 0.37 0.24 ± 0.04 2.75 ± 0.09 1.43 ± 0.14

0 day 1.62 ± 0.18 9.14 ± 0.53 0.76 ± 0.11 15.71 ± 0.52 0.62 ± 0.07 3.40 ± 0.13 13.40 ± 0.83 4.73 ± 0.55 1.78 ± 0.33 1.50 ± 0.16 17.78 ± 1.37 5.01 ± 0.40 1.96 ± 0.27 4.60 ± 0.40 1.69 ± 0.13 0.81 ± 0.04 0.32 ± 0.03 0.60 ± 0.05 0.80 ± 0.07 11.86 ± 0.53 1.16 ± 0.15 0.99 ± 0.11 4.35 ± 0.11 0.38 ± 0.07 0.04 ± 0.00 7.76 ± 0.29 0.31 ± 0.00 0.17 ± 0.00 0.25 ± 0.05 2.98 ± 0.30 3.64 ± 0.13 2.09 ± 0.11 3.63 ± 0.32 0.23 ± 0.03 3.06 ± 0.20 1.85 ± 0.15

5 days 0.39 ± 0.07 2.43 ± 0.28 0.08 ± 0.02 15.11 ± 0.91 0.10 ± 0.02 0.66 ± 0.10 10.73 ± 0.64 0.40 ± 0.06 0.82 ± 0.13 0.48 ± 0.01 11.53 ± 1.14 3.14 ± 0.39 1.35 ± 0.14 1.96 ± 0.17 1.56 ± 0.15 0.69 ± 0.04 0.22 ± 0.03 0.54 ± 0.05 0.61 ± 0.07 3.33 ± 0.42 1.10 ± 0.13 0.56 ± 0.09 3.75 ± 0.21 0.71 ± 0.08 0.06 ± 0.00 7.80 ± 0.35 0.14 ± 0.00 0.15 ± 0.00 2.09 ± 0.42 1.31 ± 0.14 5.70 ± 0.19 1.64 ± 0.10 3.67 ± 0.21 0.48 ± 0.04 3.08 ± 0.10 1.99 ± 0.08

10 days 0.08 ± 0.01 0.68 ± 0.13 0.13 ± 0.01 12.33 ± 0.88 0.08 ± 0.01 0.63 ± 0.05 7.93 ± 0.29 0.38 ± 0.06 1.50 ± 0.31 0.30 ± 0.01 9.24 ± 0.98 3.62 ± 0.33 0.73 ± 0.07 1.37 ± 0.13 0.80 ± 0.13 0.54 ± 0.06 0.21 ± 0.03 0.53 ± 0.05 0.67 ± 0.09 2.56 ± 0.30 2.17 ± 0.20 0.41 ± 0.10 3.59 ± 0.18 1.83 ± 0.16 0.03 ± 0.00 6.66 ± 0.28 0.13 ± 0.01 0.10 ± 0.00 18.11 ± 1.03 1.76 ± 0.27 3.91 ± 0.48 2.15 ± 0.07 3.43 ± 0.13 0.22 ± 0.04 2.52 ± 0.17 1.72 ± 0.13

15 days 0.05 ± 0.01 0.48 ± 0.06 0.06 ± 0.00 10.92 ± 0.55 0.03 ± 0.00 0.53 ± 0.05 7.46 ± 0.49 0.37 ± 0.12 1.22 ± 0.15 0.28 ± 0.01 8.21 ± 0.81 3.47 ± 0.36 0.47 ± 0.09 1.44 ± 0.21 0.84 ± 0.12 0.44 ± 0.06 0.31 ± 0.05 0.48 ± 0.04 0.54 ± 0.06 2.50 ± 0.28 0.74 ± 0.15 0.46 ± 0.09 3.68 ± 0.24 1.82 ± 0.21 0.00 ± 0.00 6.22 ± 0.20 0.08 ± 0.00 0.49 ± 0.09 19.86 ± 1.15 1.88 ± 0.18 4.03 ± 0.25 1.26 ± 0.10 3.34 ± 0.20 0.35 ± 0.05 1.19 ± 0.06 0.88 ± 0.07

20 days

Days after anthesis

0.07 ± 0.01 0.50 ± 0.05 0.39 ± 0.02 6.19 ± 0.37 0.07 ± 0.00 0.32 ± 0.06 5.17 ± 0.43 0.31 ± 0.03 1.62 ± 0.26 0.27 ± 0.02 7.29 ± 0.31 2.81 ± 0.20 0.27 ± 0.04 1.23 ± 0.11 0.61 ± 0.09 0.11 ± 0.01 0.33 ± 0.03 0.27 ± 0.04 0.41 ± 0.05 1.31 ± 0.33 2.89 ± 0.29 0.13 ± 0.03 1.79 ± 0.11 1.86 ± 013 0.00 ± 0.00 5.19 ± 0.35 0.08 ± 0.00 0.21 ± 0.01 12.20 ± 0.93 1.98 ± 0.17 2.27 ± 0.15 0.87 ± 0.08 1.91 ± 0.12 0.32 ± 0.04 0.51 ± 0.08 0.42 ± 0.11

25 days 0.01 ± 0.00 0.22 ± 0.01 0.21 ± 0.03 2.54 ± 0.29 0.01 ± 0.00 0.05 ± 0.00 4.08 ± 0.19 0.13 ± 0.02 0.68 ± 0.11 0.17 ± 0.01 3.61 ± 0.24 0.35 ± 0.10 0.04 ± 0.01 0.14 ± 0.03 0.15 ± 0.04 0.04 ± 0.01 0.18 ± 0.04 0.12 ± 0.01 0.33 ± 0.06 0.76 ± 0.14 0.70 ± 0.06 0.10 ± 0.02 1.03 ± 0.13 1.86 ± 0.25 0.00 ± 0.00 2.75 ± 0.24 0.07 ± 0.00 0.12 ± 0.00 5.18 ± 0.48 1.94 ± 0.29 1.34 ± 0.11 0.35 ± 0.05 0.24 ± 0.03 0.52 ± 0.07 0.46 ± 0.03 0.42 ± 0.09

30 days

0.01 ± 0.00 0.03 ± 0.00 0.12 ± 0.01 2.38 ± 0.29 0.01 ± 0.00 0.00 ± 0.00 2.10 ± 0.10 0.07 ± 0.01 0.48 ± 0.02 0.01 ± 0.00 1.78 ± 0.06 0.21 ± 0.05 0.04 ± 0.01 0.12 ± 0.00 0.06 ± 0.01 0.03 ± 0.00 0.10 ± 0.00 0.08 ± 0.00 0.11 ± 0.01 0.78 ± 0.04 0.62 ± 0.10 0.06 ± 0.01 0.76 ± 0.12 2.40 ± 0.23 0.00 ± 0.00 2.26 ± 0.25 0.06 ± 0.00 0.06 ± 0.00 2.16 ± 0.36 0.23 ± 0.04 1.09 ± 0.13 0.33 ± 0.05 0.22 ± 0.03 0.30 ± 0.04 0.43 ± 0.04 0.45 ± 0.01

35 days

Low Higha Low Higha Low Higha Higha Low Low Low Higha Higha Low Higha Low Low Low Low Low Higha Low Low Higha High Low Higha Low Low High Higha Higha Higha Higha Low Higha Higha

Expression groups

Table 1. Relative expression levels and expression groups of the genes encoding starch synthesis at daytime and degradation enzymes at the following night in wheat flag leaves during anthesis. Transcription levels of the above genes were analyzed by qPCR using Actin gene as internal control. a The transcription levels of the gene are positively and significantly related to the contents of leaf starch during anthesis.

important role in transitory starch synthesis via plastidial AGP Glc synthesis in wheat flag leaves. TaAGPS1-b and TaAGPL1, which encode the cytosolic small and LSU, respectively, of the AGPase enzyme, had similar expression profiles after anthesis in wheat flag leaves: expressed at low levels during all stages of flag leaf growth with a minimum in expression 5 days after anthesis and quickly declining thereafter before leveling off (Table 1). Clearly, the amounts of TaAGPL2 and TaAGPS2 were markedly higher than those of TaAGPS1-b and TaAGPL1 (Table 1). This implies that TaAGPS1-b and TaAGPL1 possibly associate to form a heterotetrameric cytosolic AGPase, which may play an important role in starch accumulation via AGP Glc synthesis in wheat grains. Transcripts of TaAGPS1-a, which encodes another cytosolic SSU of AGPase, were not detected in wheat flag leaves after anthesis. Granule-bound starch synthase and soluble starch synthase The starch synthase genes in wheat are divided into five branches: TaGBSS and TaSSI–SSIV. The TaGBSS, TaSSII and TaSSIII proteins are subclassified into two or three groups: TaGBSSI and TaGBSSII; TaSSIIa, TaSSIIb and TaSSIIc; and TaSSIIIa and TaSSIIIb; respectively (Jeon et al. 2010, Kang et al. 2013). The TaGBSSII gene was vigorously expressed at the beginning of anthesis (0 and 5 days after anthesis). The transcripts declined quickly to low levels 10 days later and then gradually decreased thereafter (Table 1). The transcript levels of the TaGBSSI gene were low at all stages after anthesis and its weak maximum appeared 5 days after anthesis (Table 1). During almost all stages after anthesis, transcripts of TaGBSSII were significantly higher than those of TaGBSSI. Soluble starch synthase Among all starch synthases, the number of SS isoforms was the highest (Table S2) and their transcript profiles were also highly variable (Table 1). The transcript levels of TaSSI and TaSSIIIb genes were already high at the beginning of anthesis and quickly increased to a peak 5 days after anthesis, gradually decreasing thereafter (Table 1). TaSSIV transcripts were expressed at moderate levels in the early and middle stages after anthesis, then decreased gradually 30 and 35 days after anthesis (Table 1). This suggests that TaSSI and TaSSIIIb could be the major SS forms in wheat flag leaves. TaSSIIa, TaSSIIb and TaSSIIIa had comparatively low transcript levels during all stages of flag leaf growth after 20

anthesis (Table 1). Additionally, TaSSIIc transcripts were not detected in flag leaves following anthesis. Starch branching enzyme TaBE in wheat is divided into three clades, TaBEI–III, and TaBEII is subclassified into TaBEIIa and TaBEIIb. Transcripts of TaBEIIa were abundant at the beginning of anthesis, peaked 5 days after anthesis and then quickly declined thereafter (Table 1). Both TaBEI and TaBEIII were expressed at comparatively moderate transcription levels at the beginning of anthesis, but gradually declined until leaf senescence (Table 1). In contrast, transcripts of TaBEIIa were higher than those of both TaBEI and TaBEIII at almost all stages of flag leaf growth after anthesis, suggesting that TaBEIIa might account for the major BE activities in wheat flag leaves. TaBEIIb transcripts were not detected at any stage after anthesis. Starch debranching enzyme DBE in wheat is divided into two groups, TaISA and TaPUL, and the TaISA clade is further divided into three branches: TaISA1, TaISA2 and TaISA3. TaPUL and the three TaISA genes were expressed at low levels with similar temporal profiles at all stages of flag leaf growth after anthesis (Table 1). Phosphorylase PHO in wheat was divided into two subclasses, plastidial PHOL and cytosolic PHOH. TaPHOL was highly expressed in flag leaves at the beginning of anthesis, peaked 5 days later, precipitously declined until 10 days after anthesis and then gradually decreased during subsequent stages, coinciding temporally with transitory starch accumulation in flag leaves (Table 1). This suggests that TaPHOL might play an important role in the synthesis of transitory starch in flag leaves. Transcript levels of TaPHOH were low for 10 days after anthesis, increased while fluctuating from 15 to 25 days after anthesis and decreased from 30 days after anthesis onward (Table 1). Disproportionating enzyme TaDPE in common wheat is also divided into two clades, TaDPE1 and TaDPE2, with similar expression profiles. The transcripts in flag leaves were high at the onset of anthesis, slowly increased to a peak 5 days thereafter and then dropped gradually (Table 1). The amount of TaDPE2 transcripts was consistently higher than TaDPE1 in all sampling periods (Table 1). In common wheat, 16 putative cDNA sequences of starch breakdown genes were identified (Table S3). The

Physiol. Plant. 153, 2015

expression profiles of these genes in wheat flag leaves after anthesis at 23:00 h in the following evening were also further analyzed by qPCR using the actin gene as an internal control (Table 1) and are described below. Similar results were obtained using GAPDH as another internal control (Table S4). 𝜶-Amylase Four full-length AMY cDNA (AMY1–4) have been found in higher plants (Radchuk et al. 2009), whereas AMY 2 was found in common wheat (Table S3). Of the three AMY genes, in this study, only the TaAMY3 transcript was not detected in wheat flag leaves (Table 1). Transcripts of the TaAMY4 gene were high for 20 days after anthesis and declined dramatically thereafter, but remained abundant until the end of flag leaf growth. Transcripts of TaAMY1 were low at the onset of anthesis and then gradually increased up to the level of TaAMY4 transcripts until leaf senescence (Table 1). Except at day 35 following anthesis, transcripts of the TaAMY4 gene were remarkably higher than those of the TaAYM1 gene across all sampling periods, indicating that TaAMY4 is needed in great amounts during the transitory starch degradation process in flag leaves in the dark. 𝜷-Amylase Five (TaBAM2–6) of seven BAM genes were expressed after anthesis in wheat flag leaves (Table 1), while TaBAM1 and 7 were not detected. The transcripts of TaBAM6 in wheat flag leaves were abundant at the beginning of anthesis and remained so for 20 days after, followed by a quick decline until leaf senescence (Table 1). Although the TaBAM5 transcripts were also abundant during 5 days after anthesis, they dramatically decreased at 10 days after and then thereafter slowly increased and remained at high levels until 30 days after anthesis, but quickly declined at the end of leaf growth (Table 1). TaBAM4 transcripts were rare 10 days after anthesis and then rapidly increased to a peak at 20 days after anthesis, dropping to a low but significant level until the end of anthesis (Table 1). The other TaBAM genes (i.e. TaBAM2 and TaBAM3) were barely expressed throughout all stages after anthesis (Table 1). Phosphoglucan water dikinase TaPWD transcripts in wheat flag leaves were already plentiful at the onset of anthesis and further increased to a peak at 15 days later, but then gradually decreased from 35 days after anthesis (Table 1).

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Glucan water dikinase This transcript was not detected in wheat flag leaves after anthesis. Phosphorylase TaPHOL transcripts were highly abundant at the onset of anthesis in flag leaves; then expression was abruptly downregulated, although transcripts remained detectable until the end stage of leaf growth. Transcript levels of TaPHOH were lower and constant in flag leaves at all stages after anthesis (Table 1). Disproportionating enzyme Although the absolute number of transcripts of TaDPE2 was consistently lower than TaDPE1, the expression patterns of the two genes were similar (Table 1). Their transcripts were already plentiful at the onset stage after anthesis, further increased to a peak at 5 and 10 days after anthesis for TaDPE1 and TaDPE2, respectively, and then slowly decreased thereafter. Expression profile categories of the genes encoding starch synthesis and degradation enzymes in flag leaves According to the above results, the expression profiles of wheat starch synthesis and degradation genes in flag leaves after anthesis were divided into two major groups (Table 1). The first group was characterized by high expression levels in flag leaves after anthesis at some stage of leaf growth. The genes with this pattern were TaAGPS2, TaAGPL2, TaGBSSII, TaSSI, TaSSIIIb, TaSSIV, TaBEIIa, TaPHOL and TaDPE2 for starch synthesis and TaAMY1, TaAMY4, TaBAM4, TaBAM5, TaBAM6, TaPWD, TaPHOL, TaDPE1 and TaDPE2 for starch degradation. The transcription levels of most of these genes (16/18) were significantly and positively related to the transitory starch content across all sampling periods after anthesis (Table 1). The other group, composed of TaAGPS1-a, TaAGPS1-b, TaAGPL1, TaGBSSI, TaSSIIa, TaSSIIb, TaSSIIc, TaSSIIIa, TaBEI, TaBEIIb, TaBEIII, TaISA1, TaISA2, TaISA3, TaPUL, TaPHOH and TaDPE1 for starch synthesis and TaAMY2, TaBAM1, TaBAM2, TaBAM3, TaBAM7, TaGWD and TaPHOH for starch breakdown, had basal or low transcript levels, or their transcripts were not detected in flag leaves throughout almost all stages following anthesis. However, no significant relationship was observed between the transcription levels of these genes and the transitory starch content in flag leaves across all sampling periods after anthesis (data not shown). 21

Discussion Phenotypic, physiological and ultrastructural analysis of wheat flag leaf after anthesis revealed the temporal and spatial changes in transitory starch in this tissue In many important crops (e.g. wheat, rice) during the reproductive stage, the growing grains represent a strong carbohydrate sink compared to the vegetative organs, which decreases photosynthesis and accelerates leaf senescence (Jessica et al. 2008). As shown in this study, the wheat flag leaf slowly became yellow after flowering and the chlorophyll pigment and MDA contents, which are important indicators of growth and senescence, gradually decreased and increased, respectively (Figs 1 and 2). These phenotypic and physiological changes were further confirmed by subsequent ultrastructural observations of flag leaf mesophyll cells. Chloroplasts present in photosynthetic organs are functionally important for photosynthesis and consequently also for grain yields in wheat, and their function is characterized by well-developed chloroplasts with a high proportion of grana stacks and stromal thylakoids. A close relationship exists between chloroplast ultrastructure and the capacity for transitory starch synthesis in leaves after anthesis (Kong et al. 2010). In this study, ultrastructural observations of flag leaf mesophyll cells at the end of the day (18:30 h) from 0 to 20 days after anthesis indicated that the mesophyll cells contained a large number of chloroplasts (Fig. 4A), each developing numerous grana with a high proportion of granal stacks (Fig. 3A, C, E, G, I). At these stages, many starch granules were accumulated in chloroplasts (Fig. 4B), indicating that transitory starch was abundantly synthesized. This was similar to the high rates of flag leaf photosynthesis at these stages (Lu et al. 2002). At subsequent leaf growth stages (around 25–35 days after anthesis), the thylakoid membranes of chloroplasts gradually degraded and the starch granule population rapidly declined (Figs 3K, M, O and 4B), indicating that leaf senescence had commenced, and the rates of photosynthesis and transitory starch synthesis decreased gradually (Kong et al. 2010). The change in the number of starch granules in mesophyll cells almost coincided with the high levels of transitory starch throughout all stages of the flag leaf growth period during the day of each sampling time point after anthesis (Figs 4B and 5). During the following dark period, transitory starch granules synthesized during the day are gradually degraded and these photosynthetic assimilates are directly transferred into grains to constitute the storage starch (Jeon et al. 2010). At the end of the subsequent night, microscopic observation indicated that starch 22

granules formed in the day disappeared partially or completely (Fig. 3B, D, F, H, J, L, N, P). This suggests that transitory starch was stored in flag leaf chloroplasts during the day and was broken down at the following night for export for the synthesis of storage starch in grains or to provide a source of carbon for growth (Lu et al. 2005). However, some incompletely degraded starch granules were also observed in chloroplasts at the end of the night at many sampling time points after anthesis (Figs 3H, J, L and 5), indicating that many transitory starch granules accumulated during the day in these stages could not be fully utilized and it was not the source (leaf) but the sink (grain) that was the main limiting factor in wheat grain yield. These results are similar to those in previous reports (Necolas et al. 1984, Bhullar and Jenner 1986). Thus, we speculate that storage starch content and wheat grain yields could be significantly improved if these leaf transitory starch granules were completely degraded and efficiently utilized for the synthesis of storage starch in wheat grains by enhancing the activity of starch-degrading enzymes in leaves or starch-synthesizing enzymes in grains. Plastoglobule in chloroplasts is thylakoid-associated monolayer lipoprotein particle containing some prenyl and neutral lipids and several dozen proteins, and its central role has been speculated to act as a component of a thylakoidal respiratory chain which is involved in starch degradation (Gfeller and Gibbs 1985, Lundquist et al. 2012). The number of plastoglobules increased dramatically in chloroplasts after anthesis (Figs 3 and 4C), implying that metabolite transport from transitory starch degradation in this organ may be enhanced (Singh and McNellis 2011, Lundquist et al. 2012). Roles of genes involved in transitory starch synthesis in wheat flag leaves based on expression profiles during the day after anthesis The genomes of two model plants, Arabidopsis and rice, have been sequenced completely, allowing the prediction of the total number of genes involved in starch biosynthesis; 22 and 29 cDNA sequences encoding different isoforms of starch synthesis genes have been identified in Arabidopsis and rice, respectively (Smith et al. 2004, Ohdan et al. 2005). In a previous study, after retrieving the wheat cDNA or EST sequences from the NCBI database using cDNA sequences of rice starch synthesis genes, 26 cDNA sequences corresponding to starch synthesis genes were identified in common wheat (Kang et al. 2013). This indicated that multiple isoforms of each enzyme are found in Arabidopsis, rice and wheat, some of which may be plant species-specific (Ohdan et al. 2005, Hannah and James 2008). For

Physiol. Plant. 153, 2015

example, AGPS2 was identified in rice and maize, but it is not present in Arabidopsis (Yan et al. 2009). AGPL3 and APGL4 were not detected in wheat and barley, but they are present in the genomes of rice and maize (Ohdan et al. 2005, Radchuk et al. 2009, Yan et al. 2009, Kang et al. 2013). Genetic studies provide evidence that different isoforms of starch synthesis-related enzymes probably play specific roles in determining the complex structure of starch (Jeon et al. 2010). Thus, the transcript levels of these genes expressed in the wheat flag leaf, the most important photosynthetic and transitory starch synthetic organ, were further analyzed in this study. Our results revealed two major expression patterns for the wheat starch-synthesizing genes in wheat flag leaves after anthesis. One group was characterized by high levels of expression after anthesis. The genes in this group included TaAGPS2, TaAGPL2, TaGBSSII, TaSSI, TaSSIIIb, TaSSIV, TaBEIIa, TaPHOL and TaDPE2 (Table 1). Their transcripts in the flag leaf were significantly and positively related to the level of transitory starch (Table 1), suggesting that they may act in close coordination to synthesize starch granules in this tissue. The functions of these genes in transitory starch synthesis in cereal crops, to our knowledge, have not been particularly examined. In Arabidopsis, the mutation in the gene encoding the SSU (Agps2) of leaf AGPase resulted in the near absence of starch in leaf blades (Lin et al. 1988a). Similarly, mutants (Adg2) of the gene encoding the LSU of leaf AGPase also greatly decreased the amount of leaf starch (Lin et al. 1988b). In addition, the important roles of GBSSII (Hirose and Terao 2004), SSI (Delvallé et al. 2005), SSIIIb (Zhang et al. 2005), SSIV (Crumpton-Taylor et al. 2013), BEIIa (Blauth et al. 2001) and DPE2 (Lütken et al. 2010) in the synthesis of leaf transitory starch have also been confirmed by mutant studies in Arabidopsis and rice. The function of the PHOL gene in leaf transitory starch synthesis has not been shown in higher plants, although its role in the synthesis of rice seed storage starch has already been confirmed (Satoh et al. 2008). In the other group, transcripts of the genes encoding TaAGPS1-a, TaAGPS1-b, TaAGPL1, TaGBSSI, TaSSIIa, TaSSIIb, TaSSIIc, TaSSIIIa, TaBEI, TaBEIIb, TaBEIII, TaISAI, TaISAII, TaISAIII, TaPUL, TaPHOH and TaDPE1 were low throughout the grain development period, although small maxima appeared at the beginning of anthesis (Table 1). The genes in this group might be associated with the synthesis of storage starch in the cereal endosperm. Storage starch synthesis in cereal endosperm has been studied extensively, and the distinct roles of the enzymes encoded by most of these genes in the second group have also been verified in mutants of important cereal crops including rice and wheat, exhibited by AGPS1-a (Johnson et al. 2003), AGPL1 (Bhave

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et al. 1990), GBSSI (Nakamura et al. 1995), SSIIa (Zhang et al. 2004), SSIIIa (Fujita et al. 2007), BEI (Satoh et al. 2003), BEIIb (Nishi et al. 2001), ISAI (Kubo et al. 2005), ISAII (Kubo et al. 2010), PUL (Fujita et al. 2009) and DPE1 (Tetlow et al. 2004). The functions of TaAGPS1-b, TaSSIIb, TaSSIIc, TaBEIII, TaISAIII and TaPHOH in starch synthesis in plants have not yet been elucidated. Roles of the genes involved in transitory starch breakdown in wheat flag leaves based on the expression profiles during the following night Many of the enzymes identified as belonging to the degradation pathway also have multiple isoforms. In barley, the important genes involved in transitory starch degradation were identified in the barley pericarp, another transitory starch storage tissue (Radchuk et al. 2009). Using the barley genes involved in transitory starch degradation, 16 genes involved in starch breakdown in common wheat were identified in this study (Table S3). A cDNA sequence encoding the AMY3 gene was not identified in common wheat in this study, possibly because it plays a minor role in starch breakdown in higher plants (Yu et al. 2005) or because the complete genome of common wheat has not been sequenced, although the genomes of the A-genome (Triticum urartu) and D-genome (Aegilops tauschii) progenitors of wheat were sequenced recently (Jia et al. 2013, Ling et al. 2013). Similarly, this study showed two major expression patterns for the starch-degrading genes in wheat flag leaves after anthesis during the subsequent night. The first group included TaAMY1, TaAMY4, TaBAM4, TaBAM5, TaBAM6, TaPWD, TaPHOL, TaDPE1 and TaDPE2 characterized by vigorous expression during the early or middle stages after anthesis, when transitory starch was highly synthesized in the day and quickly degraded the following night (Table 1). The expression profiles of most of these genes coincided with a decrease in starch granules (Fig. 4B; Table 1). The genes exhibiting this pattern could play crucial roles in the breakdown of transitory starch, and upregulation of these genes in the flag leaf suggests that transient starch turnover occurs in this tissue to avoid carbon starvation and optimize the supply of carbon to the developing endosperm (Smith and Stitt 2007). The coordinated expression of specific isoforms may be important for proper starch biosynthesis, because starch biosynthetic enzymes in maize and wheat have recently been shown to be associated in multisubunit complexes (Tetlow et al. 2008, Radchuk et al. 2009). The functions of BAM5 (Laby et al. 2001), GWD3 (Baunsgaard et al. 2005), PHOL, DPE1 (Critchley et al. 2001) and DPE2 (Chia et al. 2004) in the breakdown of transitory starch 23

in Arabidopsis leaves were already confirmed. AMY4 is a newly described AMY and it regulates starch breakdown, operating independently of other BAMs (Fulton et al. 2008). Its high expression in the wheat flag leaf implies that it may be the major form of the AMY enzyme having important functions in starch breakdown combined with TaBAM5 (Table 1). Little or no data are available on the function of BAM6 in other higher plant species. Moderate levels of TaBAM 6 in the early and late stages of the flag leaf suggest that it may also be involved in starch breakdown in common wheat (Table 1). The second group, composed of TaAMY1, TaAMY2, TaBAM1, TaBAM2, TaBAM3, TaBAM7 and TaPHOH, has low expression levels in wheat flag leaves at all stages after anthesis (Table 1). Minor roles for AMY1 and 2 (Stanley et al. 2002), BAM1 (Valerio et al. 2011), BAM2 (Fulton et al. 2008), BAM7 (Reinhold et al. 2011) and PHOH (Zeeman et al. 2004) in starch breakdown have been elucidated in the leaves of Arabidopsis, in which BAM3 has a role in starch breakdown (Fulton et al. 2008). However, transcripts of BAM3 were not detected in barley (Radchuk et al. 2009) and barely expressed in the wheat flag leaf (Table 1), implying that it may have weak function in starch degradation in cereal crops. Comparison of gene expression patterns in flag leaves and grains of common wheat suggests two different pathways for starch synthesis and degradation in these two tissues On the basis of the transcript profiles, nine starch synthesis-related genes (TaAGPS2, TaAGPL2, TaGBSSII, TaSSI, TaSSIIIb, TaSSIV, TaBEIIa, TaPHOL and TaDPE2) are assumed to have important functions in leaf transitory starch synthesis (Table 1). However, our previous results showed that the expression profiles of 13 starch synthesis-related genes (TaAGPS1-a, TaAGPL1, TaGBSSI, TaSSI, TaSSIIa, TaSSIIIa, TaSSIV, TaBEI, TaBEIIa, TaBEIIb, TaPUL, TaPHOL and TaDPE1) possibly play a crucial role in storage starch synthesis in wheat grains (Kang et al. 2013). Four genes, including TaSSI, TaSSIV, TaBEIIa and TaPHOL, had high expression in the flag leaves and grains of wheat plants in these two studies, indicating that they may commonly participate in transitory and storage starch synthesis in both flag leaves and grains in wheat plants. Similar results have been reported in rice, in which the SSI gene was highly expressed in both endosperm and leaf sheath (another important photosynthetic organ) and was proposed to commonly function in both transitory and storage starch synthesis in this species (Hirose et al. 2006). However, most of the above-identified starch synthesis-related genes in the flag leaves and grains of common wheat were different. The 24

Table 2. Comparisons between the transcript profiles of transitory starch synthesis-related genes in the leaf of common wheat identified in this study and in the young leaf of rice identified by Ohdan et al. (2005). Expression profiles of identified transitory starch synthesis-related in two plant species Commonly expressed starch synthesis-related genes in both rice and wheat (six genes) Specially expressed starch synthesis-related genes in the leaf of common wheat (three genes) Specially expressed starch synthesis-related genes in the leaf of rice (three genes)

Names of the identified genes AGPS2, AGPL2 or 3, GBSSII, SSIIIb, BEIIa, DPE2 TaSSI, TaSSIV, TaPHOL

OsSSIIb, OsISA3, OsPHOH

specific genes involved in transitory starch synthesis in the leaf include TaAGPS2, TaAGPL2, TaGBSSII, TaSSIIIb and TaDPE2, while TaAGPS1-a, TaAGPL1, TaGBSSI, TaSSIIa, TaSSIIIa, TaBEI, TaBEIIb, TaPUL and TaDPE1 were predominantly identified in storage starch synthesis in wheat grains (Table 1) (Kang et al. 2013). Many differences exist in the biochemical characterization between transitory and storage starches, including the shapes and sizes of starch granules, amylose content and the distribution of branch lengths in amylopectin (Tomlinson et al. 1997). For example, leaf starch granules are smaller than grain starch granules, leaf starch contains both branched glucans of high molecular weight and relatively unbranched glucans of lower molecular weight and leaf starches generally appear to have lower amylose content than storage starches (Santacruz et al. 2004). These findings imply that the above-identified genes that are solely involved in transitory or storage starch synthesis between the leaf and grain can account for the different catalytic and/or regulatory properties of each starch type. Transitory starch synthesized in the day in plant leaves is degraded during the subsequent night to supply sucrose to non-photosynthetic tissues (Lu et al. 2005, Geigenberger 2011). Accordingly, transcripts of many starch degradation-related genes were plentiful at night in the flag leaves of common wheat in this study (Table 1). However, these genes were rarely detected in non-photosynthetic tissues (including grain) in closely related species such as barley (Radchuk et al. 2009). The expression profiles of the genes encoding starch degradation enzymes may help to ensure that no significant turnover of starch occurs in the starchy endosperm during the filling period. Although transcripts of some starch degradation-related genes were also detected in crop grains, the related enzymes encoded by these genes can be inactive (Beck and

Physiol. Plant. 153, 2015

Comparison of the expression profiles of the genes involved in transitory starch metabolism between common wheat and other cereal crops

CO2

Daytime

Leaf

Photosynthesis

Glucose-6P Glucose-1P TaAGPS2

TaAGPL2

ADP-glucose TaSSIV TaBEIIa TaPHOL TaDPE2

TaGBSSII TaSSI TaSSIIIb

Transitory starch TaAMY1

TaAMY4

Night

Malto-oligo saccharides TaBAM5 TaBAM6

TaPHOL

Maltose TaDPE1

TaDPE2

Glucose

Transfusion tissue Glucose-1P TaAGPS-a

Grain

TaAGPL1

ADP-glucose TaGBSSI TaSSI TaSSIIa TaSSIIIa TaSSIV TaBEI

TaBEIIa TaBEIIb TaPUL TaPHOL TaDPE1

Storage starch

Fig. 6. Schematic figures showing the genes predominantly involved in starch synthesis and degradation in both leaf and endosperm of common wheat. Refer Kang et al. (2013) for more details of the genes expressed in endosperm of common wheat.

Ziegler 1989, Radchuk et al. 2009). These findings suggest that different pathways for starch breakdown may also exist between the leaf and grain. Transcript profiles of starch metabolism-related genes in both leaf and grain led us to propose different pathways for starch metabolism in these two tissues (Fig. 6).

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To our knowledge, transcript expression profiles of the genes encoding transitory starch synthesis-related enzymes in leaf tissues have so far been reported in two model plants, Arabidopsis and rice (Smith et al. 2004, Ohdan et al. 2005, Hirose et al. 2006). Transcript expression profiles of the genes encoding transitory starch degradation-related enzymes have not been temporally analyzed in leaf tissues of higher plants, but were measured in barley pericarp, in which the expression profiles of starch metabolism-related genes were speculated to be similar to leaves (Radchuk et al. 2009). Rice and barley are both closely related to wheat. Therefore, in this study, the transcript expression profiles of starch synthesis-related genes in the rice leaf and starch degradation-related genes in barley pericarp were compared with our findings. Many of the starch metabolism-related genes that were highly expressed in the leaves of rice or pericarp of barley were also commonly identified in this study. For example, AGPS2, AGPL2 or 3 (cDNA sequences of TaAGPL2 had 92% identity to OsTaAGPL3, and they were referred to as one gene), GBSSII, SSIIIb, BEIIa and DPE2 were commonly identified in leaves of wheat and rice and are thought to have important roles in transitory starch synthesis in these two species (Table 2). In addition, AMY1, AMY4, BAM5, BAM6, PWD and PHOL were also commonly identified in both flag leaves of wheat and the pericarps of barley, and are thought to play important roles in transitory starch breakdown in both the flag leaves of wheat and pericarp of barley (Tables 1 and 3) (Radchuk et al. 2009). These data suggest that many genes can commonly function in starch metabolism among different plant species. We also found that some starch metabolism-related genes were species-specific. For example, the TaSSI, TaSSIV and TaPHOL genes are thought to have important functions in transitory starch synthesis in wheat flag leaves, whereas OsSSIIb, OsISA3 and OsPHOH possibly function in transitory starch synthesis in rice leaves (Table 2). With respect to transitory starch degradation, TaBAM4, TaDPE1 and TaDPE2 were only identified in flag leaves of common wheat at night, while HvBAM2, HvBAM7 and HvGWD1 were found in the barley pericarp (Table 3). These indicated that some genes involved in starch metabolism are species-specific. And these differences may be due to the divergence of DNA sequences among these species (Santacruz et al. 2004, Jeon et al. 2010, Weise et al. 2011). 25

Table 3. Comparisons between the transcript profiles of transitory starch degradation-related genes in the leaf of common wheat identified in this study and in the pericarps of barley identified by Radchuk et al. (2009). Expression profiles of identified transitory starch degradation-related in two plant species Commonly expressed starch degradation-related genes in both barley and wheat (six genes) Specially expressed starch degradation-related genes in the leaf of common wheat (three genes) Specially expressed starch degradation-related genes in the pericarp of barley (three genes)

Names of the identified genes AMY1, AMY4, BAM5, BAM6, PWD, PHOL

Acknowledgements – This study was financially supported by the National Natural Science Foundation of China (31171471 and 30871472), the Special Modern Agricultural Industry (Wheat) Technology System (CARS-03), the Twelfth Five-Year National Food Production Technology Project (2011BAD16B07) and the Open Item of the State Key Laboratory of Crop Biology (2013KF04). The authors declare no competing financial interest.

TaBAM4, TaDPE1, TaDPE2

References HvBAM2, HvBAM7, HvGWD1

Conclusion Microscopic observation indicated that starch granules were synthesized in chloroplasts of mesophyll cells in wheat flag leaves the day after anthesis, while these starch granules were degraded the following night. Transitory starch synthesis in the flag leaves of common wheat was preferentially regulated by a few genes during the day (TaAGPS2, TaAGPL2, TaGBSSII, TaSSI, TaSSIIIb, TaSSIV, TaBEIIa, TaPHOL and TaDPE2), whereas transitory starch degradation in this tissue was largely regulated by the TaAMY1, TaAMY4, TaBAM4, TaBAM5, TaBAM6, TaPWD, TaPHOL, TaDPE1 and TaDPE2 genes during the following night. Comparisons of the transcript profiles of the genes encoding starch metabolism enzymes between grains and flag leaves showed that two different pathways for starch metabolism exist in these two tissues. In addition, comparison with expression profiles for leaf starch metabolism among some important cereal crops suggests that the genes involved in starch metabolism may be species-specific. This study provides specific information on the physiological and molecular mechanisms of transitory starch synthesis and degradation in the leaf of higher plants.

Author contributions G. K., X. P. and L. W. conducted the physiological and transcriptional experiments, analyzed data and wrote the manuscript; Y. Y., R. S. and Y. X. conducted the ultrastructural experiments; D. M. and C. W. participated in analysis of data and assisted in writing the manuscript; T. G. and Y. Z. designed the experiments and checked 26

the article. All authors have read and approved the final manuscript.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Meteorological parameters (rainfall, temperature, etc.) during anthesis in the experimental field. Table S2. Primer sequences of the genes encoding starch synthesis enzymes in common wheat. Table S3. Primer sequences of the genes encoding starch degradation enzymes in common wheat. Table S4. Relative expression levels and expression groups of the genes encoding starch synthesis at daytime and degradation enzymes at the following night in wheat flag leaves during anthesis. Transcription levels were analyzed by qPCR using GAPDH gene as internal control.

Edited by B. Huang

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