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TaNF-YB3 is involved in the regulation of photosynthesis genes in Triticum aestivum Troy J. Stephenson¹², C. Lynne McIntyre¹, Christopher Collet² and Gang-Ping Xue¹*

¹ CSIRO Plant Industry, 306 Carmody Road, St Lucia QLD 4067, Australia. ² Cell and Molecular Biosciences Discipline, Queensland University of Technology, GPO Box 2434, Brisbane QLD 4001, Australia. * Corresponding author Tel: 61 7 3214 2354 Fax: 61 7 3214 2920 e-mail: [email protected] e-mail addresses of other authors [email protected]; [email protected]; [email protected]

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Abstract Nuclear Factor Y (NF-Y) transcription factor is a heterotrimer comprised of three subunits: NF-YA, NF-YB and NF-YC. Each of the three subunits in plants is encoded by multiple genes with differential expression profiles, implying the functional specialisation of NF-Y subunit members in plants. In this study, we investigated the roles of NF-YB members in the light-mediated regulation of photosynthesis genes. We identified two NF-YB members from Triticum aestivum (TaNF-YB3 & 7) which were markedly upregulated by light in the leaves and seedling shoots using quantitative RT-PCR. A genome-wide coexpression analysis of multiple Affymetrix Wheat Genome Array datasets revealed that TaNF-YB3-coexpressed transcripts were highly enriched with the Gene Ontology term photosynthesis. Transgenic wheat lines constitutively overexpressing TaNF-YB3 had a significant increase in the leaf chlorophyll content, photosynthesis rate and early growth rate. Quantitative RT-PCR analysis showed that the expression levels of a number of TaNF-YB3-coexpressed transcripts were elevated in the transgenic wheat lines. The mRNA level of TaGluTR encoding glutamyltRNA reductase, which catalyses the rate limiting step of the chlorophyll biosynthesis pathway, was significantly increased in the leaves of the transgenic wheat. Significant increases in the expression level in the transgenic plant leaves were also observed for four photosynthetic apparatus genes encoding chlorophyll a/b-binding proteins (Lhca4 and Lhcb4) and photosystem I reaction center subunits (subunit K and subunit N), as well as for a gene coding for chloroplast ATP synthase  subunit. These results indicate that TaNF-YB3 is involved in the positive regulation of a number of photosynthesis genes in wheat. Keywords: NF-Y transcription factor, CCAAT-box, photosynthesis, light response, gene regulation, bread wheat.

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Introduction Light regulates a number of important physiological processes in plants, including photosynthesis to acquire energy from light (Casal and Yanovsky 2005). Jiao et al. (2005) have shown that light signalling induces differential expression of at least 20% of all Arabidopsis and rice genes. Plants sense the quality, quantity, direction and duration of light through at least four distinct families of photoreceptors: phytochromes, cryptochromes, phototropins and zeitlupes (Christie 2007; Devlin et al. 2007; Li and Yang 2007; Bae and Choi 2008). These photoreceptors trigger effectors such as transcription factors, kinases, phosphatases and degradation pathway proteins (Chen et al. 2004; Casal and Yanovsky 2005). Some experimental evidence indicates that light-mediated gene regulation may also involve the Nuclear Factor Y (NF-Y) proteins as some members of this transcription factor family are light-upregulated (Stephenson et al. 2010) and the assembly of a NF-Y complex at the promoter of a photosynthesis gene is regulated by light (Kusnetsov et al. 1999). NF-Y is a heterotrimer that is formed from NF-YA, NF-YB and NF-YC subunits and binds specifically to the CCAAT-box, a cis-acting promoter element in eukaryotes (Mantovani 1998). Whereas a single gene encodes each of the three NF-Y subunits in mammals and yeast, multiple genes encode each subunit in plants. For example, Arabidopsis NF-YA, NF-YB and NF-YC subunit families have 10, 13 and 13 members, respectively (Siefers et al. 2009) and similar sizes of NF-Y subunit families have been reported in wheat and rice (Stephenson et al. 2007; Thirumurugan et al. 2008). Many plant NF-Y subunit members show differential gene expression during development and in response to external stimuli (Gusmaroli et al. 2001, 2002; Chen et al., 2007; Stephenson et al. 2007, 2010; Warpeha et al. 2007; Li et al. 2008; Thirumurugan et al. 2008; Siefers et al. 2009; Liu and Howell 2010). Recently, some subunit members have been shown to be capable of specific interaction with other regulators in the regulation of flowering time genes and stress response genes (Wenkel et al. 2006; Cai et al. 2007; Kumimoto et al. 2008; Distelfeld et al. 2009; Yamamoto et al. 2009; Liu and Howell 2010; Kumimoto et al. 2010). These studies suggest that some NF-Y subunit members in plants have evolved a degree of functional specialisation. The involvement of NF-Y members in the regulation of photosynthesis has been implicated in a few previous studies (Miyoshi et al. 2003; Warpeha et al. 2007; Stephenson et al. 2010). In rice, antisense and RNAi constructs to OsHAP3A (NF-YB) resulted in reduced chlorophyll content in the leaves, degenerate chloroplasts and a marked reduction in the mRNA level of a light harvesting chlorophyll-a/b binding protein gene (CAB) (Miyoshi et al. 3

2003). Mutation of NF-YA5 and NF-YB9 subunit members in Arabidopsis resulted in loss of the blue light fluorescence-mediated expression of light harvesting chlorophyll a/b-binding protein (Lhcb) in etiolated seedlings (Warpeha et al. 2007). In wheat, a gene expression correlation analysis has shown that a light-upregulated TaNF-YC11 is predominantly expressed in photosynthetic organs and is closely co-regulated with a number of photosynthesis genes in various expression profiling datasets (Stephenson et al. 2010). It indicates that TaNF-YC11 may have a role in the regulation of photosynthesis genes, likely through the formation of a NF-Y heterotrimer with other light-upregulated partners and subsequent binding to light-responsive CCAAT-box cis-elements in the promoters of photosynthesis genes. The promoters of a number of photosynthesis genes, such as CAB, in wheat contain the CCAAT-box (Stephenson et al. 2010). We are interested in identifying NF-Y subunit members involved in the regulation of photosynthesis genes in wheat to gain an understanding of the gene networks that underlie carbon assimilation. This paper reports the identification of a light-upregulated NF-YB gene (TaNF-YB3) which is significantly correlated in expression with photosynthesis-related transcripts in at least three separate Affymetrix Wheat Genome Array datasets. The potential role of light-upregulated members of the plant NF-YB subunit family in the regulation of photosynthesis genes was investigated by creating transgenic wheat lines that constitutively overexpressed TaNF-YB3. This transgenic wheat study showed that overexpression of TaNFYB3 resulted in the elevated mRNA levels of a number of genes involved in photosynthesis, such as the components of photosynthesis apparatus, and enzymes involved in the chlorophyll biosynthesis pathway and light-driven ATP synthesis in chloroplasts. The overexpression transgenic data together with positive expression correlation data between TaNF-YB3 and photosynthesis genes provide experimental evidence for that TaNF-YB3 is one of the ratelimiting factors involved in the positive regulation of a number of photosynthesis genes in wheat. Furthermore, overexpression of TaNF-YB3 in transgenic wheat increased the leaf chlorophyll content, photosynthesis rate and early growth rate.

Materials and methods

Plant materials and treatments

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Spring wheat (Triticum aestivum L. cv. Bobwhite) plants were grown in a controlledenvironment growth room in 1.5-litre pots under night/day conditions of 14/18°C, 90/60% relative humidity and 16-h light with a photosynthetically active radiation flux of 500 µmol m-2 s-1 at the plant canopy level as described previously (Stephenson et al. 2010). For the light and dark treatment experiment of 23-day-old wheat plants, plants were grown with the daily 16-h light/8-h dark cycle as above. For the purpose of investigating gene expression difference in dark and light environments, 40-h dark treatment was used, which enables us to observe gene response to dark well beyond diurnal variation and with a reasonable length of time for degradation of much of the transcript that was produced during the light period. The fully expanded new leaves were collected from the plants after 6-h light exposure (i.e. 6 h after the light turned on) or after 40-h dark treatment. The daily 16-h light/8-h dark cycle for the plants with 40-h dark treatment was interrupted after a 14-h light period and plants were placed in another controlled-environment growth room at the start of the dark treatment. Leaves from both light- and dark-treated plants were sampled at a similar time point (within 30 minutes from start to end) to minimise the impact of diurnal variation in expression. For dark- and light-treatments of seedlings, wheat seeds were germinated in wet tissue paper for 5 days at 20°C in either complete darkness or continuous white fluorescent lights. At the time of sampling, the residual endosperm starch of the grains was still visible.

Construction of TaNF-YB3 expression cassette A 946-bp fragment, including the full-length coding region (639 bp) and the 5'- and 3'untranslated regions of TaNF-YB3, was amplified from wheat leaf cDNA (cv. Babax) using polymerase

chain

reaction

(PCR)

and

the

following

ACAAGTGTCCTTCCTTCCAGTTA-3’;

primers:

sense,

antisense,

5'5'-

TTCATGGAGAGCTTCCCAGGTATG-3’. The fragment was gel purified using the QIAEX II Gel Extraction Kit (QIAGEN), cloned into the vector pGEM-T easy (Promega) and sequenced (GenBank accession number HM777005). The 639-bp region encoding TaNF-YB3 was

amplified

by

PCR

using

the

primer

pair

TaNFYB3S1

CGGGATCCTAAGCAACTTAATTAAACCATGCCGGAGTCGGACAACGACT-3’) TaNFYB3A2

(5'and

(5'-GACTAGTAAGCTTACCCCTCTTTCCGTCCGAACCCCGACGAA-3’)

which also served to introduce terminal restriction sites (underlined) to facilitate directional 5

cloning. The PCR-amplified fragment was inserted into a pUbiSXR-based expression vector (Vickers et al. 2003), containing the promoter and first intron of the maize Ubiquitin-1 (Ubi1) gene and the terminator region from the rice RBCS (Christensen and Quail 1996; Matsuoka et al. 1988). The 3-kb TaNF-YB3 expression cassette was confirmed by nucleotide sequencing.

Wheat transformation Immature embryos from the wheat strain Bobwhite SH98 26 were transformed by particle bombardment as described previously (Pellegrineschi et al. 2002). The TaNF-YB3 expression cassette and selectable marker gene cassette [(rice Act1:Bar:Nos 3') constructed from pAAI1GUSR and pDM803 (Patel et al. 2000)] were amplified from expression plasmids constructed in the pSP72 vector by PCR using the following primers SP72HA2 (5'CCGAACGACCGAGCGCAGC-3’) and SP72X5 (5'-AACTATGCGGCATCAGAGCAG-3’) anchored in the vector sequence. The amplified gene cassettes were purified using the QIAquick PCR Purification Kit (QIAGEN). The TaNF-YB3 expression cassette was cobombarded with the selectable marker gene cassette into the immature embryos. The herbicide phosphinothricin was used for selection of transformed calli. Plantlets generated through the wheat transformation process were grown in a controlled-environment growth room as described above, except 16/20°C (night/day) growth temperatures were used for all experiments involving transgenic plants.

Quantitative RT-PCR analysis Total RNA was isolated, purified and converted to cDNA as described previously (Xue and Loveridge 2004). Transcript levels for the selected genes were quantified by real-time PCR as described by Stephenson et al. (2010). Gene-specific primer pairs for TaNF-YB3-coexpressed genes are listed in Supplementary Table S1. In addition to the reference genes TaRPII36 and TaRP15, a phosphoglucomutase gene (TaPGM2) was included as an internal control gene (Supplementary Table S1) (Xue et al. 2006, 2008a).

Identification of potential target genes Using Affymetrix GeneChip® data analysis and Gene Ontology enrichment analysis

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Identification of potential NF-Y target genes using Affymetrix GeneChip® data analysis and GO enrichment analyses was carried out as essentially described by Stephenson et al. (2010). Affymetrix Wheat Genome Array expression datasets were collected from EMBL-EBI ArrayExpress Browser (http://www.ebi.ac.uk/microarray-as/ae, including those initially deposited at GEO at NBCI) (Parkinson et al. 2009). The Affymetrix wheat genome array contains 61,127 probe sets representing 55,052 transcripts for all 42 chromosomes in the wheat genome. Eight Affymetrix datasets (E-MEXP-1193, E-GEOD-9767, E-MEXP-971, EGEOD-6227, E-MEXP-1523, E-GEOD-6027, E-GEOD-4935, E-GEOD-5942) were collected for analysis (Crismani et al. 2006; Jordan et al. 2007; Mott and Wang 2007; Qin et al. 2008; Wan et al. 2008; Xue et al. 2008b). The raw Affymetrix array data was normalised using a robust multi-array average using a log scale measure of expression using the default settings for the Bioconductor affy package within the R statistical programming environment (http://www.r-project.org/) as described previously (Xue et al. 2008b). The normalised expression values were converted back to non-log values for correlation analysis. Pearson correlation coefficients (r) were calculated between the mRNA levels of TaNF-YB3 and those of all other genes in each Affymetrix Wheat Genome Array dataset. Enrichment analysis was performed

using

the

AgriGO

Gene

Ontology

(GO)

enrichment

analysis

tool

(http://bioinfo.cau.edu.cn/agriGO/) (Du et al. 2010). False discovery rate adjusted P-values of ≤ 1 × 10-10 were considered to indicate highly enriched GO terms. Sequences representing probe sets significantly correlated with the expression profiles of TaNF-YB3 were collected from

the

Triticum

aestivum

Gene

Index

database

(TaGI)

(Release

12.0,

ftp://occams.dfci.harvard.edu/).

Chlorophyll content measurement Wild-type and transgenic leaves were collected and immediately immersed in liquid nitrogen. Samples were homogenised using a ball mill and 200 mg of each sample was extracted with 80% acetone. Leaf chlorophyll content was determined by measurement of absorbance at 645 and 663 nm as per the method of Yoshida et al. (1971).

Photosynthetic rate measurement Photosynthetic rate was determined by gas exchange analysis, which was performed on intact and fully expanded new leaves of 4-week-old plants using a LI-6400 portable photosynthesis 7

system (LI-6400, Li-Cor Inc., Lincoln NE, USA). The leaf to be measured was placed in a 2 × 3 cm leaf chamber with a built-in red + blue light–emitting diode light source (LI-COR 640002B) at the light setting of 500 µmol photons m–2 s–1. Photosynthesis rate was measured as the net assimilation rate of CO2 by the leaf and was expressed as mol CO2 m-2 s-1. Plants were kept in the growth room at a light level of 500 µmol photons m–2 s–1 during the gas exchange analysis.

Results

Members of the TaNF-YB subunit family are upregulated by light To identify whether TaNF-YB subunit members are differentially expressed in response to light their transcript levels were measured using quantitative RT-PCR in both 5-day-old and 23-day-old wheat plants with light or dark treatment. Among the 11 TaNF-YB members (Stephenson et al. 2007), TaNF-YB5 expression was not detectable in wheat leaves and seedling shoots. Four TaNF-YB genes (TaNF-YB3, 6, 7, & 9) were light-upregulated by more than 2-fold in the shoots of 5-day-old seedlings (Fig. 1a). TaNF-YB9 was light-upregulated by ~5-fold in the seedling shoots, but was not expressed in the leaves of 23-day-old plants (Fig. 1b). Three TaNF-YB genes (TaNF-YB3, 7 & 8) were upregulated by more than 4-fold in the leaves of 23-day-old wheat plants in response to light (Fig. 1b). In particular, the mRNA level of TaNF-YB3 in the light-treated leaves of 23-day-old plants was 17 times higher than that in the dark-treated leaves. However, only two TaNF-YB members (TaNF-YB3 and TaNF-YB7) were upregulated by more than 2-fold by light in both 5-day-old seedling shoots and 23-dayold wheat leaves.

Genes that are correlated in expression with TaNF-YB3 in large scale expression profiling datasets are enriched with those involved in photosynthesis To identify the potential target genes of NF-Y complexes containing light-upregulated TaNFYB subunit members, a genome-wide expression correlation analysis was used to find transcripts with significantly correlated expression profiles with light-upregulated TaNF-YB members. Several Affymetrix Wheat Genome Array datasets were analysed for this purpose. Of the light-upregulated TaNF-YB members, one (TaNF-YB3) had a perfectly matching probe 8

set (Ta.2879.1.S1_at), while the remaining did not have representative probe sets (Supplementary Table S2). Of the Affymetrix Wheat Genome Array datasets collected, four [E-GEOD-6027 (developing anthers), E-MEXP-1523 (heat-stressed leaves), E-MEXP-971 (salt-stressed shoots) and E-GEOD-6227 (rust-infected leaves)] were found to be suitable for expression correlation analysis, as TaNF-YB3 hybridisation signals (expression values) are in a reliable range (mean hybridisation signal values within a dataset being over 300 in these four datasets) and vary by more than two-fold within each dataset. Transcripts were selected for further analysis if significantly correlated expression was identified in a minimum of three separate datasets. Positive relationships were selected by focusing on transcripts expressed higher than their potential regulator (TaNF-YB3), based on the general assumption of signal amplification from a transcription factor to its target genes. This analysis identified 449 probe sets that were significantly correlated in expression with TaNF-YB3 in at least three datasets and had higher hybridisation signals than TaNF-YB3 on average (data not shown). To identify the potential biological roles of these TaNF-YB3-coexpressed transcripts, a functional enrichment analysis tool for crop species was used to search for enriched Gene Ontology (GO) terms. Among these TaNF-YB3-coexpressed transcripts, 407 probe sets had GO annotation with 27 GO terms being highly enriched (P-value < 1 × 10-10) (Table 1). The most enriched GO term of the biological process ontology type was photosynthesis (GO:0015979, P-value = 1.9 × 10-68, 68 probe sets) (Table 1). The most enriched GO terms of the cellular component and molecular function ontology types were chloroplast (GO:0009507, P-value = 2.5 × 10 71 , 135 probe sets) and chlorophyll binding (GO:0016168, P-value = 4.1 × 10

30

, 21 probe sets), respectively (Table 1). A list of TaNF-YB3-correlated

photosynthesis genes is shown in Table 2, which includes genes encoding the components of photosynthetic apparatus (e.g., light harvesting chlorophyll a/b-binding proteins associated with photosystem I or II, photosystem I subunits and oxygen-evolving enhancer proteins), enzymes involved in carbon fixation through the Calvin cycle (e.g., RBCS, sedoheptulose1,7-bisphosphatase and fructose-1,6-bisphosphatse) and enzymes involved in the chlorophyll biosynthetic pathway (e.g., glutamyl-tRNA reductase, chloroplast 1-deoxy-D-xylulose-5phosphate synthase and uroporphyrinogen decarboxylase). These data indicate that the role of TaNF-YB3 is potentially associated with photosynthesis. The organs used for generating these four datasets have high levels of TaNF-YB3 transcript based on Affymetrix array

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hybridisation signals and are all capable of photosynthesis, including anthers (Clément et al. 1997).

TaNF-YB3-coexpressed genes are upregulated by light in the leaf and seedling shoots and their mRNA levels are positively correlated with TaNF-YB3 To investigate whether TaNF-YB3-coexpressed transcripts identified from Affymetrix Wheat Genome Array datasets are light-upregulated and are positively correlated with TaNF-YB3 expression in the light- and dark-treated leaf and seedling shoot samples, we have examined our previously generated expression data of a set of 13 photosynthesis genes: ATP synthase B chain (TaATPa9), ATP synthase  subunit (TaATPaG), light harvesting Chlorophyll a/bbinding

proteins

(TaLhca2,

TaLhca4

and

TaLhcb4),

Fructose-1,6-bisphosphatase

(TaFBPa5), Ferredoxin-NADP(H) oxidoreductase (TaFNR), Glutamyl-tRNA reductase (TaGluTR), Oxygen-evolving enhancer protein 1 (TaOEE), Plastocyanin (TaPC), Photosystem I reaction center subunit K (TaPSIK), Photosystem I reaction center subunit N (TaPSIN) and Thioredoxin M-type (TaTRXM). As reported previously, all of these genes are light-upregulated in wheat leaves and seedling shoots (Stephenson et al., 2010). A further analysis revealed that the mRNA levels of 10 of the 13 genes (TaATPa9, TaATPaG, TaLhca2, TaLhca4, TaLhcb4, TaFBPa5, TaFNR, TaOEE, TaPSIK and TaPSIN) were significantly correlated with that of TaNF-YB3 among the leaf and seedling samples of plants with light or dark treatment (Table 3). The remaining three genes showed significantly positive correlation in expression with TaNF-YB3 in the leaves of 23-day-old plants with dark or light treatment. These positive correlation data suggest that close coregulation between TaNF-YB3 and these photosynthesis genes also occurs in the leaves of wheat plants in response to light.

Overexpression of TaNF-YB3 upregulates photosynthesis genes in Triticum aestivum To investigate whether light-upregulated TaNF-YB genes have a role in the regulation of photosynthesis genes, we produced transgenic wheat lines constitutively overexpressing TaNF-YB3 as a representative of these TaNF-YB genes. Quantitative RT-PCR was used to determine the relative transcript levels of a number of TaNF-YB3-coregulated photosynthesis genes in TaNF-YB3-overexpressing transgenic wheat plants. Transgenic wheat plants were generated by transforming wheat (cv. Bobwhite) with a TaNF-YB3 expression construct (UbiNF-YB3) driven by the maize Ubi-1 promoter. Three transgenic lines were selected for 10

molecular analysis and the presence of the transgene UbiNF-YB3 was verified by PCR using UbiNF-YB3-specific primers (Fig. 2a). The

relative transcript levels of TaNF-YB3 in

transgenic lines was determined by quantitative RT-PCR and were found to be 3-19 times higher in these three transgenic lines than in Bobwhite control plants (Fig. 2b). The differences in the TaNF-YB3 transcript levels observed between the TaNF-YB3overexpressing lines and Bobwhite were much higher than those that were found among samples in each of the four Affymetrix Wheat Genome Array datasets used for the expression correlation analyse. Therefore, these TaNF-YB3-overexpressing lines were used for analysis of potential TaNF-YB3 target genes. The transcript levels of 11 photosynthesis genes [TaLhca2, TaLhca4, TaLhcb4, TaPSIN, TaPSIK, thylakoid ascorbate peroxidase (tAPX), chloroplast ferredoxin (TaPetF), TaFBPase, sedoheptulose-1,7-bisphosphatase (TaSBPase), TaATPaG and TaGluTR] were analysed. All of these photosynthesis genes appeared to have increased expression levels in the transgenic lines, compared to Bobwhite control (Fig. 3a). TaLhca4 transcript levels were significantly higher in the leaf from all three transgenic lines than Bobwhite (Fig. 3a). TaLhcb4 transcript levels were significantly higher in two lines. In the highest TaNF-YB3 overexpressing line (UbiNFYB3-38), the expression levels of four photosynthesis genes (TaLhca4, TaLhca2, TaLhcb4 & TaATPaG) were significantly upregulated (Fig. 3a). Comparison between the mean transcript levels of all transgenic lines with Bobwhite controls showed that overexpression of TaNF-YB3 in the transgenic wheat resulted in significant increases in the mRNA levels of four photosynthesis apparatus genes (TaLhca4, TaLhcb4, TaPSIN, TaPSIK), one gene involved in the chlorophyll synthetic pathway (TaGluTR) and another gene (TaATPaG) encoding chloroplast ATP synthase  subunit (Fig. 3b).

Overexpression of TaNF-YB3 resulted in increased leaf chlorophyll content, photosynthesis and early growth rate in Triticum aestivum To further investigate the biological function of TaNF-YB3, two transgenic lines (UbiNFYB3-38 and UbiNF-YB3-46) were selected for phenotypic analysis. A striking phenotype of these transgenic lines was the visibly greener leaves of the TaNF-YB3-overexpressing lines than those of the wild-type control (Bobwhite). Therefore, the leaf chlorophyll content was measured. The total chlorophyll, chlorophyll a and chlorophyll b contents in the leaves of 4week-old wheat plants were significantly increased in the transgenic lines compared to Bobwhite (Fig. 4a-c), which appears to coincide with the increased expression level of 11

TaGluTR, encoding a known rate-limiting enzyme in the chlorophyll synthetic pathway (Tanaka and Tanaka 2006). To examine whether the increased expression of photosynthetic genes and leaf chlorophyll content has an effect on photosynthesis, the photosynthesis rates were also measured in the leaves of plants from these transgenic lines in comparison with Bobwhite. As shown in Figure 4d, the leaf photosynthesis rates per unit of leaf area were significantly higher in the transgenic lines than Bobwhite. An increase in the photosynthesis rate appears to be accompanied by an increased growth rate. Four-week-old TaNF-YB3 overexpressing lines had a significant increase in plant height and an approximately 30% increase in shoot fresh weight and dry weight compared to Bobwhite (Fig. 5a-c).

Discussion This study identified two TaNF-YB members (TaNF-YB3 and TaNF-YB7) that were lightupregulated by more than 2-fold in both the leaves and seedling shoots of wheat. TaNF-YB8 was also markedly light-upregulated in the leaf, but light-upregulation in the seeding shoots was less pronounced. The protein sequences of TaNF-YB3, TaNF-YB7 and TaNF-YB8 are highly homologous and cluster together in a neighbour-joining tree (Stephenson et al. 2007). These three genes have similar organ mRNA distribution profiles with predominant expression in the leaf, followed by the other green photosynthetic organs (young spike and developing stem) (Stephenson et al. 2007). In particular, the mRNA levels of these three TaNF-YB genes in the leaf are about 20-fold higher than those in the root (a nonphotosynthetic organ), indicating that their role is predominantly associated with photosynthetic organs. With the presence of a TaNF-YB3 probe set in the Affymetrix Wheat Genome Array and the availability of several Affymetrix Wheat Genome Array datasets we performed a genome-wide gene expression correlation analysis to identify TaNF-YB3coexpressed genes. This analysis revealed that TaNF-YB3-coexpressed genes were highly enriched with photosynthesis genes. Expression analysis of a selected set of TaNF-YB3coexpressed photosynthesis genes in wheat leaves and seedling shoots showed that these genes were also upregulated by light and were positively correlated in expression with TaNFYB3 in response to light. These data suggest a potential role of TaNF-YB3 in the positive regulation of photosynthesis genes.

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NF-Y transcription factor binds to the CCAAT-box, which is known to be one of the most common promoter elements in eukaryotes (Mantovani 1998, 1999; Testa et al. 2005; Siefers et al. 2009), suggesting that NF-Y is capable of regulating a large number of genes. Although experimental evidence on the binding of plant NF-Y transcription factor to the promoter elements of photosynthesis genes is limited, a NF-Y binding CCAAT-box has been functionally identified in the promoter of the spinach photosynthetic gene AtpC (Kusnetsov et al. 1999). Bioinformatic analysis has revealed the presence of the CCAAT-box elements in the promoters of several wheat photosynthetic genes (Stephenson et al. 2010). This transgenic study showed that overexpression of TaNF-YB3 in wheat resulted in significant increases in the expression levels of several photosynthesis genes that encode the components of the photosynthetic apparatus (TaLhca4, TaLhcb4, TaPSIN and TaPSIK), the  subunit of chloroplast ATP synthase (TaATPaG) and glutamyl-tRNA reductase (TaGluTR), an enzyme involved in the chlorophyll biosynthetic pathway. An increase in the expression of genes encoding enzymes (TaSBPase and TaFBPase) involved in the Calvin cycle was also observed, although it was not statistically significant. In particular, an increase in the TaGluTR transcript level in TaNF-YB3-overexpressing lines was accompanied by an elevated level of chlorophyll in the leaves. Chlorophylls are essential molecules in photosynthetic organisms as they are responsible for harvesting solar energy and are necessary for charge separation and electron transport within the photosystem reaction centers (Tanaka and Tanaka 2006). In photosynthetic eukaryotes, chlorophyll synthesis starts with the precursor 5-aminolevulinic acid (ALA), the availability of which is a primary controller of chlorophyll biosynthesis (Ilag et al. 1994). ALA is synthesised from glutamate by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT) (Mochizuki et al. 2010). ALA synthesis is the rate limiting step of the chlorophyll biosynthesis pathway in higher plants (Tanaka and Tanaka 2006). ALA synthesis is controlled at the GluTR reaction and is modulated at both transcriptional and post-translational levels (Goslings et al. 2004; Tanaka and Tanaka 2006; Peter and Grimm 2009; Mochizuki et al. 2010). For the transcriptional control GluTR mRNA levels have been shown to be directly correlated with ALA levels and chlorophyll biosynthesis in higher plants (Tanaka and Tanaka 2006), although this positive association is not observed in the unicellular green alga Chlamydomonas reinhardtii (Nogaj et al. 2005). An increase in the leaf chlorophyll content observed in the TaNF-YB3-overexpressing lines with an increased level of TaGluTR transcript

13

provides another line of evidence for the transcriptional regulation of GluTR being part of the regulatory mechanism in modulating chlorophyll synthesis in higher plants. These functional analysis data obtained from the overexpression transgenic wheat study provide supporting evidence for the involvement of TaNF-YB3 in the regulation of photosynthesis genes speculated on the basis of various expression data. The most significant finding of this study is that the enhanced expression levels of these photosynthesis genes in TaNF-YB3-overexpressing transgenic wheat leads to an increase in leaf chlorophyll content and photosynthesis rate. The transgenic results together with positive expression correlation data obtained from this study suggest that TaNF-YB3 is one of the rate-limiting factors involved in the positive regulation of photosynthesis genes. Over-production of TaNF-YB3 in transgenic wheat is likely to enhance the formation of the TaNF-YB3-associated NF-Y heterotrimer, based on the principle of stoichiometry. However, photosynthesis genes are likely to be regulated by multiple transcription factors, not just by the action of NF-Y and general transcriptional machinery. NF-Y in plants is likely to serve as a general promoter organizer, helps the binding of neighbouring factors and attracts coactivators as observed in mammalian and yeast systems (Testa et al., 2005). Therefore, further increase in the expression levels of these photosynthetic genes would require enhancement of other regulators. This limitation was observed in this study. The high TaNF-YB3 expression line (UbiNF-YB3-38) did not lead to further increase in the expression levels of the photosynthesis genes, compared to the low expression line (UbiNF-YB3-46). Involvement of NF-YB members in the regulation of some photosynthesis genes has also been shown in rice by suppression of OsHAP3A-C genes (Miyoshi et al. 2003). Suppression of OsHAP3A, OsHAP3B and OsHAP3C in transgenic rice led to degenerate chloroplasts and a significant reduction in the expression of CAB (Miyoshi et al. 2003). However, whether these three rice NF-YB genes are upregulated by light and have an influence on the expression of genes encoding enzymes such as GluTR involved in chlorophyll biosynthesis and other components of photosynthetic apparatus (e.g. photosystem I reaction center subunits) have not been reported. OsHAP3A, OsHAP3B and OsHAP3C appear to be expressed uniformly in all organs examined, including the non-photosynthetic organ root in rice (Miyoshi et al. 2003). Comparing the OsHAP3A-C amino acid sequences with those from wheat shows that they do not cluster phylogenetically with TaNF-YB3; rather OsHAP3A clusters with TaNF-YB4 and TaNF-YB10, OsHAP3B with TaNF-YB2 and OsHAP3C with TaNF-YB11 (Stephenson et al. 2007). In Arabidopsis, AtNF-YB9 has been implicated in the blue light mediated regulation of Lhcb, as the blue light induction of Lhcb 14

expression in etiolated seedlings is lost in an AtNF-YB9 mutant line generated through TDNA insertion (Warpeha et al. 2007). AtNF-YB9 (also known as LEC1) is expressed mainly within seeds during early and late embryogenesis and its expression is not detectable in the leaf under light conditions (Lotan et al. 1998; Gusmaroli et al. 2001). Unlike AtNF-YB9, the mRNA levels of TaNF-YB3 in wheat grains (endosperm and embryo) are very low, being at least 20 times lower than that in the leaf (Stephenson et al. 2007). AtNF-YB9 protein sequence clusters phylogenetically with TaNF-YB1 and TaNF-YB9 and not with TaNF-YB3 (Stephenson et al. 2007). The above expression profile and phylogenetic comparisons suggest that TaNF-YB3 is a novel member of the NF-YB subunit family with a role in the lightmediated regulation of photosynthetic genes in plants. The increased growth rate of TaNF-YB3-overexpressing lines observed at the early vegetative stage is a noteworthy phenotype. The increased growth rate in TaNF-YB3overexpressing lines is likely attributed to the enhanced photosynthesis rate observed in these transgenic lines. The fast early growth rate has also been observed in transgenic tobacco plants overproducing sedoheptulose-1,7-bisphosphatase, which leads to the enhanced photosynthesis (Lefebvre et al. 2005). An increase in the expression of the  subunit of chloroplast ATP synthase observed in TaNF-YB3-overexpressing lines may also contribute to the energy required for the increased carbon assimilation. Chloroplast ATP synthase comprises five subunits (, , ,  and ) and catalyses the light-driven synthesis of ATP coupled with an electrochemical gradient of photons established by the photoelectron transfer chain. The  subunit is considered to be important in the regulation of ATP synthase activity (Inohara et al. 1991). However, the possibility of TaNF-YB3 that is also involved in the regulation of other growth-related genes can not be excluded. The fast early growth rate can be considered as an early vigour trait, which is a physiological attribute that can enhance water-use efficiency and yield of wheat crops grown in Mediterranean-type climates via rapid canopy closure (Richards 2000, Soltani and Galeshi 2002; Mir-Mahmoodi and Soleimanzadeh 2009). Rapid canopy closure of young wheat plants reduces water loss due to evaporation from soil and hence enhances water-use efficiency (Richards 2000). Photosynthesis is a multistep physiological process, encompassing harvesting solar energy, transferring excitation energy, energy conversion, electron transfer from water to NADP+, ATP generation and a series of enzymatic reactions for assimilation of carbon dioxide (Tanaka and Makino 2009). Therefore, photosynthesis involves a large number of genes in higher plants. Photosynthetic capacity is considered to be one of the main limiting

15

factors for further improvement of the yield potential of wheat (Reynolds et al. 2009) and other grain crops (Zhu et al. 2010). Results from this study provide substantial evidence that a light-upregulated member from the NF-YB family (TaNF-YB3) is involved in the positive regulation of photosynthesis genes in wheat. The expression level of TaNF-YB3 in wheat appears to be one of the rate limiting factors in the expression of a number of photosynthesis genes. These results provide experimental evidence on possible genetic manipulation for simultaneous enhancement of several photosynthetic components to achieve the improved photosynthetic capacity of crop species in the future.

Supplementary Data Supplementary Table S1. Real-Time PCR primers of Triticum aestivum NF-YB3-correlated genes and reference genes. Supplementary Table S2. TaNF-YB members in Affymetrix GeneChip® Wheat Genome Array.

Acknowledgements This work was supported by the Australian Grains Research & Development Corporation. We would like to thank Dr. Maryse Bourgault for her help in measurement of photosynthetic rate.

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Table 1. Enriched GO terms within TaNF-YB3-correlated probe sets. GO term enrichment analysis was performed using the AgriGO Gene Ontology enrichment analysis tool (http://bioinfo.cau.edu.cn/agriGO/). GO ID indicates the Gene Ontology Identifier. Ontology types are the aspects of analysis: P = Biological Process, F = Molecular Function and C = Cellular Process. Term is the description associated with each GO ID. No. of genes indicates the number of genes within each probe set list with the associated term. A false discovery rate (FDR) adjusted P-value cut-off of 1.0 × 10-10 has been used for this table. GO ID

Ontology type

GO:0015979 GO:0019684 GO:0009765 GO:0018298 GO:0006091

P P P P P

GO:0019253

P

GO:0019685 GO:0016168 GO:0009507 GO:0009579 GO:0034357 GO:0009521 GO:0009522 GO:0009536 GO:0009534 GO:0031976 GO:0044434 GO:0031984 GO:0044435 GO:0044436 GO:0009523 GO:0042651 GO:0055035 GO:0009535 GO:0031090 GO:0044446 GO:0044422

P F C C C C C C C C C C C C C C C C C C C

Term photosynthesis photosynthesis, light reaction photosynthesis, light harvesting protein-chromophore linkage generation of precursor metabolites and energy reductive pentose-phosphate cycle photosynthesis, dark reaction chlorophyll binding chloroplast thylakoid photosynthetic membrane photosystem photosystem I plastid chloroplast thylakoid plastid thylakoid chloroplast part organelle subcompartment plastid part thylakoid part photosystem II thylakoid membrane plastid thylakoid membrane chloroplast thylakoid membrane organelle membrane intracellular organelle part organelle part

No. of Genes 68 39 27 20 50

FDRadjusted Pvalue 1.9 × 10-68 7.6 × 10-47 1.8 × 10-34 3.0 × 10-26 1.9 × 10-19

14

1.0 × 10-15

14 21 135 78 69 45 32 223 45 45 58 45 58 45 31 38 36 36 47 80 80

7.2 × 10-15 4.1 × 10-30 2.5 × 10-71 1.3 × 10-70 2.8 × 10-68 1.1 × 10-55 1.8 × 10-47 5.6 × 10-45 1.2 × 10-40 3.3 × 10-40 3.3 × 10-40 1.4 × 10-39 6.1 × 10-39 1.0 × 10-38 6.8 × 10-38 1.6 × 10-34 1.4 × 10-33 1.4 × 10-33 5.5 × 10-16 7.8 × 10-12 8.2 × 10-12

22

Table 2. Photosynthesis-related transcripts correlated with the mRNA levels of TaNF-YB3 in Affymetrix Wheat Genome Array datasets. Pearson correlation coefficients (r) were calculated for the expression profiles of all probe sets compared to Ta.2879.1.S1_at (TaNF-YB3) in four Affymetrix datasets: developing anthers (E-GEOD6027); salt-stressed shoots (E-MEXP-971); heat-stressed leaves (E-MEXP-1523); and rust-infected leaves (EGEOD-6227). Affymetrix IDs are the unique Affymetrix probe set identifiers. TaGI IDs are the Triticum aestivum Gene Index identifiers for contigs most likely represented by each probe set. Statistical significance of each r value was calculated using a t-distribution. Statistical significance of correlations is indicated by triple asterisks (P  0.001), double asterisks (P  0.01) and a single asterisk (P  0.05) Affymetrix ID

TaGI ID

Annotation

Developing anthers

Heatstessed leaf

Saltstressed shoot

Rustinfected leaf

Ta.22831.1.S1_x_at

TC388871

Photosystem I subunit O (PsaO)

0.97***

0.43**

0.75***

0.74***

Ta.29587.2.S1_x_at

TC429086

0.66***

0.58***

0.56***

0.91***

Ta.25600.1.S1_x_at

TC386108

Light harvesting chlorophyll a/b-binding protein associated with photosystem II (Lhcb) Lhcb1.4 Lhcb6

0.8***

0.64***

0.92***

0.81***

Ta.22984.2.S1_x_at

TC369171

Lhcb1.5

0.84***

0.81***

0.15

0.81***

Ta.27751.2.S1_x_at

TC370039

Lhcb2.2

0.95***

0.56***

0.37*

0.8***

Ta.27751.6.S1_at

TC402022

Lhcb2.1

0.91***

0.88***

0.75***

0.83***

Ta.27751.7.A1_x_at

TC454951

Lhcb1.3

0.96***

0.46**

0.78***

0.73***

Ta.3795.1.S1_x_at

TC372540

Lhcb1.5

0.81***

0.7***

0.27

0.81***

Ta.1130.1.S1_a_at

TC452191

Lhcb3

0.95***

0.55***

-0.53**

0.8***

Ta.1130.3.S1_x_at

TC413836

Lhcb3

0.93***

0.72***

0.33*

0.78***

Ta.20639.1.S1_x_at

TC378917

0.73***

0.78***

0.84***

0.62***

Ta.20639.2.A1_a_at

TC372628

Light harvesting chlorophyll a/b-binding protein associated with photosystem I (Lhca) Lhca4 Lhca4

0.95***

0.72***

0.75***

0.44**

Ta.20639.3.S1_a_at

TC382396

Lhca4

0.54**

0.84***

0.87***

0.46**

Ta.20639.3.S1_x_at

TC382396

Lhca4

0.54**

0.83***

0.87***

0.49**

Ta.2402.3.S1_x_at

TC375338

Lhca1

0.94***

0.78***

0.93***

0.79***

Ta.28496.1.A1_at

TC379848

Lhcb1.4

0.75***

0.51**

0.74***

0.83***

Ta.28496.1.A1_x_at

TC379848

Lhcb1.4

0.76***

0.51**

0.69***

0.83***

Ta.30702.1.S1_x_at

TC374577

Lhcb1.5

0.79***

0.6***

0.43**

0.8***

Ta.3249.2.S1_x_at

CK215785

Lhcb1.4

0.91***

0.82***

0.62***

0.74***

Ta.4346.1.A1_x_at

TC371215

Lhcb1.5

0.75***

0.7***

0.85***

0.84***

Ta.3366.1.S1_at

TC404327

0.87***

0.05

0.61***

0.85***

Ta.12565.2.S1_at

TC400992

Chloroplast 1-deoxy-D-xylulose-5-phosphate synthase Chloroplast-localized Ptr ToxA-binding protein1

0.76***

0.54***

0.91***

0.41**

Ta.28806.1.S1_at

TC370684

Ferredoxin-NADP(H) oxidoreductase

0.77***

-0.05

0.93***

0.82***

Ta.23273.1.S1_at

TC434552

Ferredoxin-thioredoxin reductase

-0.53**

0.59***

0.78***

0.76***

Ta.439.1.S1_at

TC368647

Chloroplast Fructose-1,6-bisphosphatase

0.72***

0.4*

0.92***

0.69***

Ta.1364.1.S1_at

TC379591

Geranylgeranyl hydrogenase

0.87***

0.36*

0.81***

0.78***

Ta.3243.1.S1_at

TC385481

Glutamyl-tRNA reductase 1

0.71***

0.82***

0.82***

0.88***

Ta.3243.1.S1_x_at

TC385481

Glutamyl-tRNA reductase 1

0.69***

0.84***

0.83***

0.87***

Ta.30808.1.S1_s_at

CK211832

0.87***

0.41**

0.92***

0.79***

Ta.1135.1.S1_at

TC376922

0.88***

0.18

0.93***

0.55***

Ta.20911.1.A1_at

TC415515

Chloroplast glyceraldehyde-3-phosphate dehydrogenase A Chloroplast glyceraldehyde-3-phosphate dehydrogenase B Kinase binding protein

0.91***

0.26

0.97***

0.85***

Ta.20911.2.A1_at

TC415515

Kinase binding protein

0.83***

0.38*

0.97***

0.84***

Ta.28697.3.S1_at

TC371257

Lhca2

0.86***

0.5**

0.92***

0.69***

Ta.30727.1.S1_at

TC381159

Lhcb4

0.96***

0.53***

0.82***

0.76***

Ta.881.1.S1_a_at

CK213344

Lhca5

0.85***

0.34*

0.95***

0.85***

23

Table 2 (continued) Affymetrix ID

TaGI ID

Annotation

Developing anthers

Heatstessed leaf

Saltstressed shoot

Rustinfected leaf

Ta.881.2.S1_x_at

TC374543

Lhca5

0.75***

0.27

0.95***

0.87***

Ta.1395.1.S1_at

TC388241

0.89***

0.65***

0.77***

0.73***

Ta.9574.1.S1_at

TC375499

Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase Mg-chelatase subunit XANTHA-F

0.91***

0.3*

0.75***

0.85***

Ta.595.1.S1_at

TC370409

Chloroplast omega-6 fatty acid desaturase

0.86***

-0.01

0.71***

0.89***

TaAffx.53766.1.S1_x_at

CA689372

Oxygen-evolving complex (Fragment)

-0.3

0.64***

0.91***

0.64***

Ta.841.1.S1_a_at

BJ319092

Oxygen-evolving enhancer protein 1

0.82***

0.68***

0.79***

0.5***

Ta.85.1.S1_at

TC372246

Oxygen-evolving enhancer protein 2

0.87***

0.58***

0.79***

0.52***

Ta.2307.1.S1_at

TC386668

Chloroplast phosphoglycerate kinase

0.82***

0.53***

0.91***

0.81***

Ta.23158.1.S1_at

TC377328

Chloroplast phosphoribulokinase

0.87***

0

0.86***

0.79***

Ta.24304.2.S1_a_at

TC456410

Photosystem I reaction center subunit II

0.86***

0.69***

0.78***

0.65***

Ta.3581.1.S1_x_at

TC369833

Photosystem I reaction center subunit III,

0.82***

0.82***

0.9***

0.62***

Ta.3581.3.S1_x_at

TC421201

Photosystem I reaction center subunit III,

0.84***

0.83***

0.9***

0.73***

Ta.1969.1.S1_a_at

TC375307

Photosystem I reaction center subunit IV

0.98***

0.38*

0.65***

0.78***

Ta.27761.1.S1_x_at

TC388041

Photosystem I reaction center subunit psaK

0.96***

0.68***

0.82***

0.78***

Ta.27761.2.S1_x_at

TC412122

Photosystem I reaction center subunit psaK

0.75***

0.69***

0.72***

0.8***

Ta.27761.3.S1_x_at

TC388041

Photosystem I reaction center subunit psaK

0.9***

0.72***

0.82***

0.77***

Ta.27751.3.S1_x_at

TC382472

Photosystem I reaction center subunit XI

0.76***

0.64***

0.74***

0.67***

Ta.27751.3.S1_at

TC373665

Photosystem I reaction center subunit XI

0.69***

0.73***

0.83***

0.65***

Ta.28363.3.S1_x_at

TC418745

0.94***

0.72***

0.95***

0.73***

Ta.1161.1.S1_at

TC375393

Photosystem I reaction centre subunit N (PSI-N) Photosystem II subunit PsbS

0.86***

0.22

0.81***

0.85***

Ta.8027.1.S1_at

TC389636

0.66***

0.52**

0.94***

0.7***

Ta.8027.1.S1_x_at

TC389636

0.69***

0.45**

0.93***

0.71***

Ta.22648.1.S1_a_at

TC398802

Chloroplast plastid-lipid-associated protein 3 Chloroplast plastid-lipid-associated protein 3 Oxygen evolving enhancer 3 (PsbQ)

0.83***

-0.19

0.95***

0.82***

TaAffx.7419.1.A1_x_at

CK216646

Ribose-5-phosphate isomerase

0.52**

0.44**

0.93***

0.85***

Ta.2752.2.S1_x_at

TC370099

0.82***

0.25

0.93***

0.52***

Ta.2752.3.S1_x_at

TC370099

Ribulose bisphosphate carboxylase small chain (rbcS) rbcS

0.8***

0.24

0.93***

0.54***

TaAffx.449.1.A1_at

CA722056

rbcS

0.53**

0.63***

0.92***

0.78***

TaAffx.108219.1.S1_at

CA690622

rbcS

0.72***

0.62***

0.95***

0.5***

Ta.1988.1.S1_x_at

TC387438

Sedoheptulose-1,7-bisphosphatase

0.76***

0.52**

0.95***

0.75***

Ta.1988.2.S1_x_at

TC373708

Sedoheptulose-1,7-bisphosphatase

0.9***

0.4*

0.97***

0.78***

Ta.1988.3.S1_x_at

TC395470

Sedoheptulose-1,7-bisphosphatase

0.87***

0.46**

0.95***

0.81***

Ta.636.1.S1_s_at

TC384854

0.74***

0.08

0.97***

0.83***

Ta.12190.1.A1_at

TC381309

0.94***

0.69***

0.97***

0.89***

Ta.23411.1.S1_at

TC376508

Thylakoid membrane phosphoprotein 14 kDa Thylakoid membrane phosphoprotein 14kDa Chloroplast transketolase

0.87***

0.18

0.79***

0.81***

Ta.4315.1.S1_at

TC392505

Chloroplast Triosephosphate isomerase

0.7***

0.05

0.79***

0.81***

Ta.27646.1.S1_at

TC454548

Lhca3

0.8***

0.77***

0.81***

0.69***

Ta.27646.1.S1_x_at

TC432666

Lhca3

0.84***

0.77***

0.68***

0.69***

Ta.28648.1.S1_s_at

TC419769

Uroporphyrinogen decarboxylase

0.96***

0.52**

0.41*

0.9***

24

Table 3. Expression correlations of photosynthesis genes with TaNF-YB3 in wheat leaves and seedling shoots with dark or light treatment. Gene ID represents identifiers based on annotation of similar sequences from the model organisms Arabidopsis and rice. TaGI represents the Triticum aestivum Gene Index identifiers. r values represent Pearson correlation coefficients between the mRNA levels of TaNF-YB3 and its co-regulated genes. Statistical significance of each r value was calculated using a t-distribution. Statistical significance of correlations is indicated by triple asterisks (P  0.001), double asterisks (P  0.01) and a single asterisk (P  0.05) Gene ID TaGI

Correlation coefficient (r) Leaf Seedling

TaATPa9 TaATPaG TaLhca4 TaFBPase TaFNR TaGluTR TaLhcb4 TaLhca2 TaOEE TaPC TaPSIK TaPSIN TaTRXM

0.96** 0.95** 0.97** 0.94** 0.94** 0.95** 0.97** 0.94** 0.91* 0.95** 0.97** 0.97** 0.98***

TC416524 TC372375 TC378917 TC368647 TC401602 TC385481 TC381159 TC371257 TC380359 CA598047 TC388041 TC385047 TC447076

0.93** 0.93** 0.97** 0.96** 0.95** 0.28 0.91* 0.93** 0.91* 0.78 0.94** 0.96** 0.47

25

Figure legends Figure 1. Changes in the mRNA levels of wheat NF-YB genes in the leaf and seedling shoot in response to dark or light treatment. (a) In the shoots of 5-day-old seedlings. (b) In the leaves of 23-day-old plants. Transcript level is expressed relative to the dark treatment. Values are means + SD of three biological samples. Each sample was analysed with triplicate PCR assays. Statistical significance of differences was analysed using the Students t-test and is indicated by triple asterisks (P  0.001), double asterisks (P  0.01) and a single asterisk (P  0.05). The sequences of these TaNF-YB genes were documented in Stephenson et al. (2007). Figure 2. Overexpression of TaNF-YB3 in transgenic wheat lines carrying the UbiNF-YB3 transgene. (a) The PCR amplification of the UbiNF-YB3 transgene in transgenic lines (at the T2 stage): UbiNF-YB3-38 (the lane labelled as 38), UbiNF-YB3-46 (the lane labelled as 46) and UbiNF-YB3-89 (the lane labelled as 89). The UbiNF-YB3 DNA fragment was amplified from genomic DNA by PCR using UbiNF-YB3-specific primers. The specificity of the UbiNF-YB3 PCR product was verified using Bobwhite (BW) as negative control, where no transgene product was amplified. M indicates lanes containing the Promega 1kb+ DNA ladder. (b) Expression levels of TaNF-YB3 in Bobwhite (BW) and T2 transgenic lines, determined by quantitative RT-PCR using real-time PCR primers that amplify both endogenous and transgene TaNF-YB3 transcripts. Expression levels are expressed relative to Bobwhite. Values are means + SD of three biological samples. Each sample was analysed with triplicate PCR assays. Statistical significance of differences was analysed using the Students t-test and is indicated by triple asterisks (P  0.001) and double asterisks (P  0.01). Figure 3. Expression levels of photosynthesis genes in Bobwhite controls and TaNF-YB3overexpressing transgenic wheat lines. (a) Expression levels of photosynthesis genes in the leaves of individual T2 transgenic lines. (b) Mean expression levels of photosynthesis-related genes in the leaves of three transgenic lines. Values are means + SD of three biological samples. All quantitative RT-PCR assays were done with three technical replicates. Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) and a single asterisk (P  0.05). Accession identifiers of these genes are listed in Supplementary Table S1. Figure 4. The increased leaf chlorophyll content and photosynthetic rate in T2 TaNF-YB3 overexpressing transgenic wheat lines. (a) Total leaf chlorophyll content. (b) Leaf chlorophyll a content. (c) Leaf chlorophyll b content. (d) Leaf photosynthetic rate per unit of leaf area. Plants at four weeks old were used for analysis. All values are the mean + SD of four biological samples. Controls are Bobwhite. Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) or a single asterisk (P  0.05). FW, fresh weight; BW, Bobwhite. Figure 5. The increased early growth rate in T2 TaNF-YB3 overexpressing transgenic wheat lines. (a) Plant height. (b) Fresh weight of shoots per plant. (c) Dry weight of shoots per plant. Plants at four weeks old were used for analysis. All values are the mean + SD of six biological samples. Controls are Bobwhite (BW). Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) or a single asterisk (P  0.05).

26

a A

TaNF-YB expression levels in the seedling shoot in response to light

** 5 4

**

***

*** Dark

3

Light

*** 2

***

1

*

B b

TaNF-YB11

TaNF-YB10

TaNF-YB9

TaNF-YB8

TaNF-YB7

TaNF-YB6

TaNF-YB4

TaNF-YB3

TaNF-YB2

0

TaNF-YB1

Relative expression levels

6

TaNF-YB expression levels in the leaf in response to light

Relative expression levels

25

*** 20

***

15 Dark Light

10

*** 5

**

**

** TaNF-YB11

TaNF-YB10

TaNF-YB9

TaNF-YB8

TaNF-YB7

TaNF-YB6

TaNF-YB4

TaNF-YB3

TaNF-YB2

TaNF-YB1

0

Figure 1. Changes in the mRNA levels of wheat NF-YB genes in the leaf and seedling shoot in response to dark or light treatment. (a) In the shoots of 5-day-old seedlings. (b) In the leaves of 23-day-old plants. Transcript level is expressed relative to the dark treatment. Values are means + SD of three biological samples. Each sample was analysed with triplicate PCR assays. Statistical significance of differences was analysed using the Students t-test and is indicated by triple asterisks (P  0.001), double asterisks (P  0.01) and a single asterisk (P  0.05). The sequences of these TaNF-YB genes were documented in Stephenson et al. (2007).

27

a

M

BW

38

46 89

M

1000 850 650 500 400 300 200

b Relative expression level

***

***

**

BW trl n o C

UbiNFYB3-38

UbiNFYB3-46

UbiNFYB3-89

Figure 2. Overexpression of TaNF-YB3 in transgenic wheat lines carrying the UbiNF-YB3 transgene. (a) The PCR amplification of the UbiNF-YB3 transgene in transgenic lines (at the T2 stage): UbiNF-YB3-38 (the lane labelled as 38), UbiNF-YB3-46 (the lane labelled as 46) and UbiNF-YB3-89 (the lane labelled as 89). The UbiNF-YB3 DNA fragment was amplified from genomic DNA by PCR using UbiNF-YB3-specific primers. The specificity of the UbiNF-YB3 PCR product was verified using Bobwhite (BW) as negative control, where no transgene product was amplified. M indicates lanes containing the Promega 1kb+ DNA ladder. (b) Expression levels of TaNF-YB3 in Bobwhite (BW) and T2 transgenic lines, determined by quantitative RT-PCR using real-time PCR primers that amplify both endogenous and transgene TaNF-YB3 transcripts. Expression levels are expressed relative to Bobwhite. Values are means + SD of three biological samples. Each sample was analysed with triplicate PCR assays. Statistical significance of differences was analysed using the Students t-test and is indicated by triple asterisks (P  0.001) and double asterisks (P  0.01).

28

a

Control UbiNF-YB3-38

4.0

UbiNF-YB3-46

3.5

UbiNF-YB3-89

3.0 **

2.5 2.0

* **

*

*

*

*

1.5

**

1.0 0.5

b

TaATPaG

TaGluTR

TaPSIK

Control

2.5

2.0

TaSBPase

TaFBPase

TaLhcb4

TaPetF

tAPX

TaLhca2

TaPSIN

TaLhca4

0.0

UbiNF-YB

*

*

*

*

* *

1.5

1.0

TaATPaG

TaGluTR

TaPSIK

TaSBPase

TaFBPase

TaLhcb4

TaPetF

tAPX

TaLhca2

TaPSIN

0.5

TaLhca4

Relative expression levels Relative expression levels

4.5

Photosynthesis genes Figure 3. Expression levels of photosynthesis genes in Bobwhite controls and TaNF-YB3overexpressing transgenic wheat lines. (a) Expression levels of photosynthesis genes in the leaves of individual T2 transgenic lines. (b) Mean expression levels of photosynthesis-related genes in the leaves of three transgenic lines. Values are means + SD of three biological samples. All quantitative RT-PCR assays were done with three technical replicates. Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) and a single asterisk (P  0.05). Accession identifiers of these genes are listed in Supplementary Table S1.

29

Chlorophyll content (mg g-1 FW)

1.65

a

**

**

1.55 1.45 1.35 1.25

BW

Chlorophyll a content (mg g-1 FW)

1.3

b

* *

1.2 1.1 1.0 0.9 0.8

BW

Chlorophyll b content (mg g-1 FW)

UbiNF-YB-38 UbiNF-YB3-46

0.45

UbiNF-YB-38 UbiNF-YB3-46

c

**

0.40

0.35

0.30 BW

Photosynthetic rate (mol CO2 m-2 s-1)

20

UbiNF-YB-38 UbiNF-YB3-46

*

d

*

16

12

8

4

0 BW

UbiNF-YB3-38 UbiNF-YB3-46

Figure 4. The increased leaf chlorophyll content and photosynthetic rate in T2 TaNF-YB3 overexpressing transgenic wheat lines. (a) Total leaf chlorophyll content. (b) Leaf chlorophyll a content. (c) Leaf chlorophyll b content. (d) Leaf photosynthetic rate per unit of leaf area. Plants at four weeks old were used for analysis. All values are the mean + SD of four biological samples. Controls are Bobwhite. Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) or a single asterisk (P  0.05). FW, fresh weight; BW, Bobwhite.

30

Plant height (mm)

460

a

** *

445 430 415 400

BW 3.0

b

UbiNF-YB3-38 UbiNF-YB3-46 **

Fresh weight (g)

* 2.8 2.5 2.3 2.0

BW

Dry weight (g)

0.45

c

0.40

UbiNF-YB3-38 UbiNF-YB3-46 **

**

**

**

0.35 0.30 0.25

BW

UbiNF-YB3-38 UbiNF-YB3-46

Figure 5. The increased early growth rate in T2 TaNF-YB3 overexpressing transgenic wheat lines. (a) Plant height. (b) Fresh weight of shoots per plant. (c) Dry weight of shoots per plant. Plants at four weeks old were used for analysis. All values are the mean + SD of six biological samples. Controls are Bobwhite (BW). Statistical significance of differences was analysed using the Students t-test and is indicated by double asterisks (P  0.01) or a single asterisk (P  0.05).

31