Characterization of Rubisco activase genes in maize: an α-isoform gene functions alongside a β-isoform gene.

Characterization of Rubisco Activase Genes in Maize: An a-Isoform Gene Functions alongside a b-Isoform Gene1[W][OPEN] Zhitong Yin 2, Zhenliang Zhang 2, Dexiang Deng, Maoni Chao, Qingsong Gao, Yijun Wang, Zefeng Yang, Yunlong Bian, Derong Hao, and Chenwu Xu* Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China (Z.Yi., Z.Z., D.D., Q.G., Y.W., Z.Ya., Y.B., C.X.); National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China (M.C.); and Jiangsu Yanjiang Institute of Agricultural Sciences, Nantong 226541, China (D.H.) ORCID ID: 0000-0003-2482-8681 (C.X.).

Rubisco activase (RCA) catalyzes the activation of Rubisco in vivo and plays a crucial role in regulating plant growth. In maize (Zea mays), only b-form RCA genes have been cloned and characterized. In this study, a genome-wide survey revealed the presence of an a-form RCA gene and a b-form RCA gene in the maize genome, herein referred to as ZmRCAa and ZmRCAb, respectively. An analysis of genomic DNA and complementary DNA sequences suggested that alternative splicing of the ZmRCAb precursor mRNA (premRNA) at its 39 untranslated region could produce two distinctive ZmRCAb transcripts. Analyses by electrophoresis and matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry showed that ZmRCAa and ZmRCAb encode larger and smaller polypeptides of approximately 46 and 43 kD, respectively. Transcriptional analyses demonstrated that the expression levels of both ZmRCAa and ZmRCAb were higher in leaves and during grain filling and that expression followed a specific cyclic day/night pattern. In 123 maize inbred lines with extensive genetic diversity, the transcript abundance and protein expression levels of these two RCA genes were positively correlated with grain yield. Additionally, both genes demonstrated a similar correlation with grain yield compared with three C4 photosynthesis genes. Our data suggest that, in addition to the b-form RCA-encoding gene, the a-form RCA-encoding gene also contributes to the synthesis of RCA in maize and support the hypothesis that RCA genes may play an important role in determining maize productivity.

Rubisco is the primary regulatory enzyme of photosynthesis and initiates photosynthetic carbon metabolism by combining atmospheric CO2 with ribulose 1,5-bisphosphate to form 3-phosphoglyceric acid. Numerous studies have shown that the activity of Rubisco is regulated by a protein known as Rubisco activase (RCA; Portis, 2003). RCA is a soluble chloroplast ATPase associated with a variety of cellular activities 1

This work was supported by the National Basic Research Program of China (grant no. 2011CB100106 to C.X.), the National Natural Science Foundation of China (grant nos. 30971846 and 31171187 to C.X.), the Vital Project of Natural Science of Universities in Jiangsu Province (grant no. 09KJA210002 to C.X. and grant no. 11KJA210004 to Z.Yi.), the Priority Academic Program Development of Jiangsu Higher Education Institutions (to Z.Yi.), and the Innovation of Science and Technology Development Fund of Yangzhou University (grant no. 2013CXJ049 to Z.Yi.). 2 These authors contributed equally to the article. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chenwu Xu ([email protected]). [W] The online version of this article contains Web-only data. [OPEN] Articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.113.230854 2096

(ATPases associated with diverse cellular activities) that functions as a molecular chaperone (Sánchez de Jiménez et al., 1995). This protein catalyzes the activation of Rubisco in vivo by the ATP-dependent removal of various inhibitory sugar phosphates (Portis et al., 2008). The activity of RCA is dependent on the ATP/ADP ratio (Zhang and Portis, 1999; Carmo-Silva and Salvucci, 2013) and/or the redox state of the chloroplast (Zhang et al., 2001; Wang and Portis, 2006) and is extremely sensitive to high temperature (Salvucci and Crafts-Brandner, 2004). Thus, RCA can adjust the rate of CO2 fixation to the rate of electron transport activity and can limit CO2 assimilation during heat stress. In many plants, there are two forms of RCA: an a isoform of 45 to 46 kD and a b isoform of 41 to 43 kD. However, in some species, such as tobacco (Nicotiana tabacum), cucumber (Cucumis sativus), and mung bean (Vigna radiata), the a isoform is believed to be absent (Portis, 2003). The greatest difference between the two forms of RCA is usually at the carboxy terminus (Salvucci et al., 1987; Portis, 2003). Compared with the b isoform, the a isoform has a carboxy-terminal extension that contains redox-sensitive Cys residues (Zhang and Portis, 1999; Portis, 2003; Salvucci et al., 2003). Both the a and b isoforms are capable of activating Rubisco;

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Rubisco Activase Genes in Maize

however, they have slightly different maximal activities (Shen et al., 1991). In rice (Oryza sativa), the a isoform has been shown to play an important role in photosynthetic acclimation to moderate heat stress in vivo, whereas the b isoform has been shown to play a major role in maintaining the initial activity of Rubisco under normal conditions (Wang et al., 2010). More significantly, light modulation of Rubisco in Arabidopsis (Arabidopsis thaliana) requires a capacity for redox regulation of the a isoform via thioredoxin-f (Zhang and Portis, 1999; Zhang et al., 2001, 2002). Genomic analyses have identified one RCA gene in spinach (Spinacia oleracea), Arabidopsis, rice, and wheat (Triticum aestivum; Werneke et al., 1988; To et al., 1999; Law and Crafts-Brandner, 2001), two RCA genes in barley (Hordeum vulgare) and cotton (Gossypium hirsutum; Rundle and Zielinski, 1991; Salvucci et al., 2003), and more than three RCA genes in tobacco (Qian and Rodermel, 1993) and soybean (Glycine max; Yin et al., 2010). In some species, such as spinach, Arabidopsis, and rice, alternative splicing of RCA transcripts results in two isoforms of RCA (Werneke et al., 1989; Rundle and Zielinski, 1991; To et al., 1999), whereas in other species such as cotton and soybean, the two RCA isoforms are encoded by different genes (Salvucci et al., 2003; Yin et al., 2010). In barley, in addition to one alternatively spliced RCA gene (rcaA) that produces two RCA isoforms, a second gene (rcaB) encodes only the b isoform of RCA (Rundle and Zielinski, 1991). Additionally, in some species such as tobacco, bean (Phaseolus vulgaris), cucumber, and mung bean, the RCA gene may only encode the b isoform of RCA (Portis, 2003). The expression patterns of the RCA gene in plants have been extensively examined. The RCA gene is expressed almost entirely in the green parts of the plant in most plant species and is developmentally regulated by leaf age and light (Watillon et al., 1993; Liu et al., 1996). In tomatoes (Solanum lycopersicum), apples (Malus domestica), Arabidopsis, and rice, the mRNA levels of the RCA gene showed cyclic variations during the day/night period (Martino-Catt and Ort, 1992; Watillon et al., 1993; Liu et al., 1996; To et al., 1999). Diversity in gene expression is one of the mechanisms underlying phenotypic diversity among individuals. In soybean, positive correlations were observed between the expression levels of two RCA genes and the initial activity of Rubisco, photosynthetic rate, and grain yield in a recombinant inbred line population, and expression quantitative trait loci (eQTL) mapping revealed four trans-eQTL for the two genes (Yin et al., 2010). Maize (Zea mays), a C4 plant, is one of the most important crops in the world, serving as an essential source of food, feed, and fuel. Various research groups have detected different numbers of RCA polypeptides in this species, varying from one to three; the molecular masses of these polypeptides are approximately 41, 43, and/or 45 to 46 kD (Salvucci et al., 1987; CraftsBrandner and Salvucci, 2002; Vargas-Suárez et al.,

2004; Ristic et al., 2009). Two RCA complementary DNAs (cDNAs), Zmrca1 and Zmrca2, have been cloned (Ayala-Ochoa et al., 2004). These two cDNAs contain identical open reading frames (ORFs) that encode the 43-kD RCA polypeptide. Based on sequence similarity, this polypeptide appears to correspond to the b isoform of RCA, as reported for other species (Werneke et al., 1989; To et al., 1999; Salvucci et al., 2003). Limited proteolysis of the 43-kD RCA at its amino-terminal region can give rise to a 41-kD RCA (Vargas-Suárez et al., 2004). Although informative, these data do not clarify the origin of the 45- to 46-kD RCA polypeptide in maize. Because the entire genome of the maize inbred line B73 has been sequenced (Schnable et al., 2009), it is possible to identify maize RCA genes on a genomewide scale. In this study, an a-form RCA gene and a b-form RCA gene were identified in the maize genome on the basis of currently available genomic resources, and the relevance of these two genes to maize RCA polypeptides was examined. In addition, the expression patterns of these two genes were investigated. Lastly, the potential relationship between these two genes and grain yield was analyzed in 123 maize inbred lines with extensive genetic variation. Our results indicate that an a-form RCA-encoding gene functions alongside a b-form RCA-encoding gene in maize and that both genes play a role in determining maize productivity.

RESULTS Genomic Analysis and cDNA Cloning Reveal a-Form and b-Form RCA Genes

In this study, a genome-wide survey of maize RCA genes was performed. The Arabidopsis RCA gene sequence information (GenBank accession no. 818558) was used to query the maize genome sequence database (http://www.phytozome.net/). We identified an a-form RCA-encoding gene and a b-form RCAencoding gene on chromosome 4, designated ZmRCAa and ZmRCAb, respectively. The identity of these two genes was corroborated by sequencing the PCR products amplified from the genomic DNA of four randomly selected inbred lines using gene-specific primers (data not shown). Two b-form RCA cDNAs, Zmrca1 (GenBank accession no. AF084478) and Zmrca2 (GenBank accession no. AF305876), were cloned and characterized in maize (Ayala-Ochoa et al., 2004). These two cDNAs contain identical ORFs but have differing 39 untranslated regions (UTRs) with different downstream-like elements. To investigate the relationship between the two b-form cDNAs and the ZmRCAb identified in this study, we cloned the genomic DNA and full-length cDNAs of Zmrca1 and Zmrca2 from the inbred line JB using genespecific primers (Supplemental Table S1). Alignment analysis (http://www.ncbi.nlm.nih.gov/spidey/) showed

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that both the Zmrca1 and Zmrca2 cDNAs precisely matched the genomic sequences of ZmRCAb, suggesting that the Zmrca1 and Zmrca2 mRNAs could arise from alternative splicing of the ZmRCAb genomic DNA (Supplemental Fig. S1). When two splice sites at the 39 UTRs were utilized, a single 112-nucleotide intron was removed to produce the Zmrca2 mRNA. Alternatively, the first 28 nucleotides of this intron were retained to produce the Zmrca1 mRNA. Sequence alignment of the Zmrca1 and Zmrca2 cDNAs identified from the maize variety Chalqueño (Ayala-Ochoa et al., 2004) and the ZmRCAb genomic DNA from the inbred line B73 (http://www.phytozome.net/) also produced the same result. Based on the genomic DNA sequence information, the cDNA of ZmRCAa was also cloned from the inbred line JB. This ZmRCAa cDNA had not been previously cloned in maize, and we deposited its sequence in GenBank under the accession number JX863889. This cDNA is predicted to encode a protein of 463 amino acids with a calculated molecular mass of 51.04 kD (Fig. 1). The first 50 amino acids at the amino terminus of ZmRCAa were predicted to be chloroplast transit peptides (ChloroP version 1.1 server; http://www.cbs. dtu.dk/services/ChloroP/). Thus, the predicted mature protein encoded by ZmRCAa contains 413 amino acids and has a calculated molecular mass of 45.96 kD. Compared with ZmRCAb, the deduced protein sequence of ZmRCAa contains a 30-amino acid extension at the carboxy terminus (Fig. 1), including two Cys residues that are known to be involved in redox regulation (Zhang and Portis, 1999; Salvucci et al., 2003).

Similar to the ZmRCAb protein, two conserved ATPbinding domains, GGKGQGKS and LFIND (Shen and Ogren, 1992), were also identified in the ZmRCAa protein. Based on its size and its long carboxy terminal region, the ZmRCAa protein appears to correspond to the a-form RCA reported in other species (Werneke et al., 1989; To et al., 1999; Salvucci et al., 2003). We also cloned the cDNAs of ZmRCAa from three other maize inbred lines, XD053, Y6, and Y53, and obtained similar results. A sequence alignment of the ZmRCAa ORFs from the different inbred lines is shown in Supplemental Figure S2.

ZmRCAa and ZmRCAb Encode Two Different Maize RCA Polypeptides

Protein extracts from Escherichia coli transformed with pET-30a expressing the truncated ORF of either ZmRCAa or ZmRCAb and several other protein extracts used as controls were separated using SDS-PAGE and probed with polyclonal cotton RCA antibodies (AS10700, Agrisera; Fig. 2A). Two polypeptides of approximately 47 and 43 kD were detected in the positive control, Arabidopsis (lane 6), which is consistent with previous findings (Salvucci et al., 1987), whereas the negative controls showed no specific bands (lanes 4 and 5). Both the ZmRCAa and ZmRCAb recombinant proteins were recognized by the anti-cotton RCA antibody (lanes 2 and 3). As expected, the molecular mass of the recombinant His-tag ZmRCAa was larger than that of the recombinant His-tag ZmRCAb. Because the two

Figure 1. Alignment of the amino acid sequences deduced from the ZmRCAa and ZmRCAb cDNAs. The sequences were aligned using the ClustalX program (version 1.81) and viewed using the GeneDOC program (version 2.6). The numbers to the right of the alignment indicate the amino acid positions in the ZmRCAa or ZmRCAb putative protein sequences. Identical amino acids (single-letter code) are shown in white characters on a black background. The arrow indicates the putative cleavage site of the putative transit peptide. The numbers in the middle of the sequences indicate the two conserved ATPbinding domains (1 and 2). The diamonds indicate the conserved Cys residues involved in redox regulation in ZmRCAa. The dashes represent gaps that were introduced into ZmRCAa or ZmRCAb to optimize the alignment. The black line and the double lines indicate the sequences used to develop the polyclonal antibodies against ZmRCAa and ZmRCAb. 2098




Figure 2. Immunological detection of RCA polypeptides. Protein extracts were separated using SDS-PAGE and probed with an anti-cotton RCA antibody. Immunoreactive proteins were detected by chemiluminescence using an anti-rabbit IgG-HRP conjugate. A, Protein markers (lane 1), recombinant His-tag ZmRCAa expressed in E. coli using the vector pET-30a (lane 2), recombinant His-tag ZmRCAb expressed in E. coli using the vector pET-30a (lane 3), untransformed E. coli (lane 4), E. coli transformed with the empty pET-30a vector (lane 5), and protein extracts from Arabidopsis leaves (lane 6). B, Protein markers (lane 1), purified maize leaf ZmRCAb (lane 2), purified maize leaf ZmRCAa (lane 3), and protein extracts from maize leaves (lane 4). The purified maize leaf ZmRCAa and ZmRCAb were prepared by immunoaffinity chromatography using their specific antibodies.

recombinant proteins contain amino-terminal His-tags, their molecular masses were larger than those of the RCA polypeptides detected in the Arabidopsis leaves (lane 6). These results confirm that ZmRCAa and ZmRCAb do encode RCA proteins. To examine the relationship of ZmRCAa and ZmRCAb to maize RCA polypeptides, two synthesized antigen peptides corresponding to the predicted sequences of ZmRCAa and ZmRCAb, respectively (Fig. 1), were used to generate specific polyclonal antibodies. In two independent western-blot experiments, both antibodies reacted with one maize leaf RCA polypeptide (data not shown). We purified maize leaf ZmRCAa and ZmRCAb using the two specific antibodies. The purified maize leaf RCAs and maize leaf protein extracts were probed with polyclonal cotton RCA antibodies (Fig. 2B). Both the purified leaf ZmRCAa and ZmRCAb showed specific bands (lanes 2 and 3). Three polypeptides were detected in maize (lane 4). The two larger polypeptides had molecular masses similar to those of the purified leaf ZmRCAa and ZmRCAb, suggesting that the maize polypeptides might correspond to the a- and b-RCA isoforms. The smallest maize polypeptide appeared to be a degradation product, possibly from proteolysis. Different numbers of RCA polypeptides, varying from one

to three, were previously detected in maize (Salvucci et al., 1987; Crafts-Brandner and Salvucci, 2002; Vargas-Suárez et al., 2004; Ristic et al., 2009). The variability in these observations might be due to the use of different affinities of the antibodies, different experimental conditions, and/or proteolysis (Salvucci et al., 1993; To et al., 1999). We used matrix-assisted laser desorption/ionization (MALDI)-tandem time-of-flight (TOF) mass spectrometry (MS) analysis to precisely determine the molecular masses of maize RCA proteins. As shown in Supplemental Figure S3, the molecular masses of purified recombinant His-tag ZmRCAa, purified recombinant His-tag ZmRCAb, purified leaf ZmRCAa, and purified leaf ZmRCAb were 52.8, 50.1, 46.1, and 43.3 kD, respectively. The determined molecular mass of the purified leaf ZmRCAa was 0.14 kD larger than its predicted molecular mass. This deviation is within the error limits (60.2 kD) of the MALDI-TOF instrument. Together with the observations made by SDS-PAGE analysis (Fig. 2), these results show that, similar to the previously characterized b-form gene ZmRCAb (Ayala-Ochoa et al., 2004; Vargas-Suárez et al., 2004), the a-form gene ZmRCAa also encodes an RCA polypeptide, but a larger one.

ZmRCAa and ZmRCAb Transcripts Are Predominantly Expressed in Leaves and Show Cyclic Day/Night Expression Patterns

At the 16-leaf stage, samples of the roots, stems, leaves, tassel spikelets, and immature ears of the maize plants were collected, and the transcript abundance of ZmRCAa and ZmRCAb in these tissues was investigated using semiquantitative reverse transcription (RT)-PCR (Supplemental Fig. S4A) and real-time quantitative RT-PCR assays (Fig. 3A). Because alternative splicing of the ZmRCAb precursor RNA (premRNA) at its 39 UTR created two transcripts with identical ORFs as mentioned above, we measured the expression of this gene by designing primers that detect the two transcripts simultaneously (Supplemental Table S1). The ZmRCAa and ZmRCAb transcripts accumulated primarily in the leaves and, to a lesser extent, in the stems, tassel spikelets, and immature ears of the plants, whereas no or very low signal was detected in the roots. We also examined the protein expression levels of ZmRCAa and ZmRCAb and obtained a similar result (Fig. 3B). At 32 d after anthesis (DAA), the leaves closest to the ear were harvested at 4-h intervals during a 48-h span from maize plants grown under a 12-hlight/12-h-dark cycle (Fig. 3C). Both semiquantitative RT-PCR (Supplemental Fig. S4B) and real-time quantitative RT-PCR (Fig. 3D) assays showed that transcripts of ZmRCAa and ZmRCAb gradually increased during the dark period, with relatively higher levels detected at 8:30 AM, 2.5 h after the beginning of the light period. Following this peak in expression, the



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Figure 3. Expression profiles of ZmRCAa and ZmRCAb in different maize tissues (A and B) and during day/night cycles (C and E). A and D, Transcript expression level measured by real-time quantitative RT-PCR. B and E, Protein expression level determined by an ELISA quantitative sandwich technique using specific antibodies. C, The 12-h-light and 12-h-dark periods over 2 d of leaf sampling are represented by white and black bars, respectively. At the 16-leaf stage, the indicated samples of the inbred line JB at 32 DAA were used to determine the expression profiles. The error bars represent the SEs of three independent repetitions.

levels of these transcripts gradually declined, reaching relatively lower levels at 8:30 PM, 2.5 h after the end of the light period. Similar results were observed previously for the two b-form RCA transcripts (Ayala-Ochoa et al., 2004). By contrast, no abrupt changes in the protein expression levels of ZmRCAa and ZmRCAb within the 12-h-light/12-h-dark periods were observed (Fig. 3E).

The Expression Levels of ZmRCAa and ZmRCAb Are Correlated with Grain Yield in Maize

The transcript abundance of ZmRCAa and ZmRCAb was determined in maize plants at various stages of growth. Both semiquantitative RT-PCR (Supplemental Fig. S4C) and real-time quantitative RT-PCR (Fig. 4A) assays showed that the ZmRCAa and ZmRCAb mRNAs 2100

were expressed at a relatively low level in the early growth stages of maize (up to tasselling) but accumulated to higher levels during the grain-filling stages (16–40 DAA), with maximum values at approximately 32 DAA. Measurement of the protein expression levels of ZmRCAa and ZmRCAb showed that the proteins encoded by the two genes were expressed at higher levels during the grain-filling stages (Fig. 4B). Because grain filling is crucial for grain formation, the higher expression levels of ZmRCAa and ZmRCAb during this stage suggested that these two genes might play a role in determining grain yield. To further investigate the relationship between the two RCA genes ZmRCAa and ZmRCAb and grain yield, we determined the transcript abundance and protein expression levels of these two genes, as well as the grain yield, in 123 maize inbred lines. Pedigree information revealed that these lines are genetically Plant Physiol. Vol. 164, 2014



2010). The transcript abundance of these genes was also significantly correlated with grain yield (Table I), and ZmRCAa and ZmRCAb expression showed a similar correlation.

DISCUSSION An a-Form Large RCA Polypeptide Gene Is Expressed in Maize

Figure 4. Expression profiles of ZmRCAa and ZmRCAb at different growth stages. A, Transcript expression level measured by real-time quantitative RT-PCR. B, Protein expression level determined by an ELISA quantitative sandwich technique using specific antibodies. The mature upper third leaves of the inbred line JB were collected at the indicated growth stages for expression profiling. The error bars represent the SEs of three independent repetitions.

diverse and that they originated from diverse germplasm resources that are currently used in China (e.g. Lancaster, Reid, Tangsipingtou, P, Lvdahonggu, and Waxy maize; Supplemental Table S2). All of the measured traits varied widely among the different inbred lines (Supplemental Tables S2 and S3). The transcript abundance of ZmRCAa and ZmRCAb in the 123 maize lines was positively correlated with their protein expression levels, and both the transcript abundance and protein expression levels of these two genes were significantly correlated with grain yield (Table I). In the 123 maize lines, we also determined the transcript abundance of three C4 photosynthesis genes, Nicotinamide Adenine Dinucleotide Phosphate-Malic Enzyme (NADP-ME), Pyruvate Orthophosphate Dikinase (PPDK), and Phosphoenolpyruvate Carboxylase (PEPC), which are widely thought to play an import role in determining grain yield (Hibberd et al., 2008; Hibberd and Covshoff,

In maize under nonstress conditions, only b-form RCA polypeptides were previously detected by westernblot analysis (Salvucci et al., 1987; Vargas-Suárez et al., 2004). In this study, we used different antibodies than those used previously, i.e. an antibody against cotton RCA and a ZmRCAa-specific antibody, and detected an a-form RCA polypeptide of approximately 46 kD (Fig. 2; Supplemental Fig. S3). By contrast, when we used an antibody against Arabidopsis RCA (aA-18; sc15864; Santa Cruz Biotechnology), we did not detect this a-form RCA polypeptide but only a polypeptide of approximately 43 kD, possibly a b-form RCA (data not shown). This variability might be due to the different affinities of the antibodies employed. Heat stress conditions might induce the expression of the a-form RCA in maize. During heat stress, a large RCA polypeptide of 45 to 46 kD, possibly the a-form RCA, was detected in maize by western-blot analysis (Sánchez de Jiménez et al., 1995; Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009). In previous studies, two b-form maize RCA cDNAs that encode the same polypeptide but show different sequences at their 39 UTRs were cloned and characterized (Ayala-Ochoa et al., 2004; Vargas-Suárez et al., 2004). In this study, on the basis of the genomic sequence of the inbred line B73, we cloned an a-form RCA gene, ZmRCAa, of maize for the first time. PCR amplification and sequence analysis confirmed that this gene is present in the genomes of diverse maize inbred lines, and both transcript and protein expression measurements showed that it is expressed in maize. The presence of an a-form RCA gene sequence in maize genome was also inferred in a recent study (Carmo-Silva and Salvucci, 2013). Similar to the phenomenon previously observed for b-form RCA genes (Ayala-Ochoa et al., 2004), ZmRCAa transcripts also show cyclic variations related to day/night period (Fig. 3), consistent with the reported involvement of the circadian clock in the accumulation of RCA mRNA (Pilgrim and McClung, 1993; Watillon et al., 1993). However, compared with the b-form RCA gene ZmRCAb, the transcript and protein expression levels of ZmRCAa were approximately 10 and 2 times lower, on average, in the 123 inbred lines (Supplemental Tables S2 and S3), indicating that the a-form RCA is not as abundant as the b-form RCA in maize under nonstress conditions. Under heat stress conditions in maize, limited proteolysis of the 43-kD b-form RCA gene product(s)



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Table I. Correlation coefficients among the transcript abundance of ZmRCAa, ZmRCAb, NADP-ME, PPDK, and PEPC, the protein expression levels of ZmRCAa and ZmRCAb, and grain yield in 123 maize inbred lines Single and double asterisks represent significance at P , 0.05 and P , 0.01, respectively. Traits

ZmRCAb Transcript Abundance

ZmRCAa Transcript Abundance

ZmRCAa transcript abundance ZmRCAb protein expression level ZmRCAa protein expression level NADP-ME transcript abundance PPDK transcript abundance PEPC transcript abundance Grain yield

0.360** 0.599** 0.364** 0.493** 0.750** 0.391** 0.489**

0.214* 0.634** 0.487** 0.408** 0.406** 0.214*

ZmRCAb Protein Expression Level

ZmRCAa Protein Expression Level

0.339** 0.273** 0.390** 0.244** 0.299**

0.377** 0.366** 0.310** 0.222*

occurs at its amino-terminal region, resulting in a smaller RCA polypeptide of 41 kD (Vargas-Suárez et al., 2004). However, no information regarding the origin of the larger heat-induced maize RCA polypeptide of 45 to 46 kD (Sánchez de Jiménez et al., 1995; Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009) is available. The polypeptide encoded by the a-form RCA gene ZmRCAa identified in this study is larger than the 43-kD polypeptide encoded by the ZmRCAb gene (Fig. 2); its molecular mass is 46.1 kD, as determined by MALDI-TOF-MS analysis (Supplemental Fig. S3). It is thus very possible that this gene contributes to the synthesis of the larger heat-induced RCA polypeptide in maize. Additional studies are required to investigate the potential role of ZmRCAa in the heat stress response of maize. The regulation of Rubisco activity by light depends on the response of RCA to the chloroplast redox state and/or the ADP/ATP ratio (Zhang and Portis, 1999; Zhang et al., 2002; Carmo-Silva and Salvucci, 2013). RCA sensitivity is conferred by two Cys residues in the carboxy terminus of the a-isoform (Zhang et al., 2002). The gene product of ZmRCAa contains these two Cys residues (Fig. 1), suggesting that this protein could play a role in the light modulation of Rubisco in maize. However, Rubisco in maize is not extensively regulated in response to changes in light intensity (Sage and Seemann, 1993), a finding that might be due to the low expression level of the a-form gene in maize, as mentioned above. Additionally, we cannot exclude the possibility that the regulatory properties of the a-form RCA in maize differ from those of RCA in other species. The activities of b-form RCAs from different species have been reported to vary when the proteins are subjected to the same physiological ratios of ADP/ATP (Carmo-Silva and Salvucci, 2013); a similar situation might also apply to the RCA a-isoform.

Posttranscriptional Regulation of the RCA Gene

Posttranscriptional regulation of gene expression occurs at the levels of premRNA processing (capping, splicing, and polyadenylation) and/or mRNA translation. In many species, alternative splicing of one 2102

NADP-ME Transcript Abundance

PPDK Transcript Abundance

0.706** 0.874** 0.328**

0.594** 0.427**

PEPC Transcript Abundance

0.306**

premRNA can produce a- and b-form RCA transcripts (Portis, 2003), whereas in soybean and cotton, the two types of transcripts arise from separate genes (Salvucci et al., 2003; Yin et al., 2010). Additionally, under heat stress conditions, RCA gene transcripts can be regulated by alternative polyadenylation of their 39 UTR (DeRidder and Salvucci, 2007). Although a previous study suggested that the two b-form RCA cDNAs Zmrca1 and Zmrca2 arise from separate genes in the maize genome (Ayala-Ochoa et al., 2004), in the light of the new evidence, knowledge, and technologies available, our study shows that these two cDNAs appear to arise from alternative splicing of the same gene. The alternative splicing sites are located in the 39 UTR of the ZmRCAb premRNA (Supplemental Fig. S1); thus, splicing at these sites would not change the amino acid sequence of the resulting protein products. Alternative splicing of premRNA at its 39 UTR is not a rare phenomenon in plants. In Arabidopsis, it was reported that approximately 6.4% of all alternativesplicing events occur in 39 UTRs (Reddy, 2007). To the best of our knowledge, this study is the first to show that alternative splicing can produce two different b-form RCA transcripts. Although it does not alter the encoded amino acid sequence, alternative splicing in the 39 UTR can produce transcripts with different sequence lengths and/or structures. In a previous study, under nonstress conditions, the Zmrca1 and Zmrca2 transcripts showed cyclic variations during a day/night period, with the Zmrca2 transcript exhibiting greater amplitude in its steady-state levels than Zmrca1 (Ayala-Ochoa et al., 2004). The authors of that study suggested that the downstream-like elements in the 39 UTRs may regulate the expression of these two transcripts. In this study, we further observed that Zmrca2 was more highly expressed than Zmrca1 in six randomly selected maize inbred lines under nonstress conditions (data not shown). In cotton and Arabidopsis plants acclimated to heat stress, stabilization of the RCA transcript levels was linked to the production of transcripts with shorter 39 UTRs (DeRidder and Salvucci, 2007; DeRidder et al., 2012). The Zmrca1 transcript has a shorter 39 UTR than the Zmrca2 transcript. Future studies aimed at investigating whether heat stress conditions induce Zmrca1 transcript expression are necessary. Plant Physiol. Vol. 164, 2014



Genome Duplication Could Potentially Be Related to the Origin of Two RCA Genes in Maize

In most plants studied to date, only one single alternatively spliced RCA gene has been identified (Werneke et al., 1988; To et al., 1999; Portis, 2003). In this study, two RCA genes, including an a-form and a b-form, were identified in the maize genome. Polyploidy is a crucial force in plant evolution, and many angiosperms have undergone one or more polyploidization events (Adams and Wendel, 2005). Compared with old polyploid plant species such as rice and Arabidopsis, which contain only one RCA gene (Werneke et al., 1989; To et al., 1999), maize experienced a recent genome duplication 5 to 12 million years ago (Schnable et al., 2011). Thus, we guess that this recent genome duplication could potentially be related to the origin of the two RCA genes identified in maize. Consistent with this idea, cotton and soybean, the two polyploid species that have most recently undergone genome duplication within the past 5 million years (Adams and Wendel, 2005), also contain at least two RCA genes (Salvucci et al., 2003; Yin et al., 2010).

The Expression Levels of ZmRCAa and ZmRCAb Could Modulate Grain Yield in Maize

By transforming the C4 plant Flaveria bidentis with an antisense RCA gene, von Caemmerer et al. (2005) showed that RCA activity was essential for the proper functioning of the C4 photosynthetic pathway. In maize, which is also a C4 plant, the RCA protein content of leaves during grain filling was higher in high-yield populations than in low-yield populations (Martı’nez-Barajas et al., 1997; Morales et al., 1999). In this study, we used a new strategy to investigate the effect of RCA on maize grain yield by determining both the transcript abundance and protein expression levels of two RCA genes in diverse maize inbred lines. The two RCA genes ZmRCAa and ZmRCAb were more highly expressed at both the transcript and protein levels during grain filling than at any other growth stages (Fig. 4; Supplemental Fig. S4). The transcript abundance and protein expression levels of these genes during grain filling were positively correlated with grain yield in 123 inbred lines (Table I). Additionally, both genes demonstrated a similar correlation with grain yield compared with three C4 photosynthesis genes. Lastly, our preliminary eQTL analysis revealed that each of the two RCA genes had one eQTL that coincided with the quantitative trait loci for grain yield in the 123 inbred lines (Supplemental Table S4). These data support the hypothesis that RCA genes play an important role in determining plant productivity. A similar result was observed in our recent studies in soybean, in which the transcript abundance of two RCA genes was positively correlated with grain yield (Yin et al., 2010; Chao et al., 2014).

It was recently reported that RCA plays an important role in regulating non-steady-state photosynthesis (Yamori et al., 2012). Both the maize in this study and the soybean in the previous studies (Yin et al., 2010; Chao et al., 2014) were grown under natural conditions and thus were exposed to a highly variable light environment each day. Plants with higher levels of RCA gene expression are thought to exhibit a more rapid increase in photosynthesis following an increase in light intensity, which could have resulted in increased grain yield. However, considering that the two RCA genes ZmRCAa and ZmRCAb showed similar correlations with grain yield as the three C4 photosynthesis genes NADP-ME, PPDK, and PEPC (Table I), it is more likely that the relationship between RCA gene expression and grain yield is a general response reflecting greater photosynthetic capacity than that it involves a more rapid increase in photosynthesis during light transients.

CONCLUSION

In maize, an a-form RCA gene, ZmRCAa, functions alongside a b-form RCA gene, ZmRCAb. ZmRCAa encodes a larger maize RCA polypeptide. Similar to ZmRCAb, ZmRCAa transcripts accumulate to higher levels in leaves than in other tissues and show cyclic variation during a day/night period. Both ZmRCAa and ZmRCAb may play important roles in determining maize productivity.

MATERIALS AND METHODS Plant Material and Plant Growth Conditions The following four maize (Zea mays) inbred lines, which have different genetic backgrounds, were used for cloning, western blotting, and/or expression pattern analysis of RCA: JB, XD053, Y6, and Y53. These four inbred lines are all members of the popular heterotic groups used in China. JB and Y53 are the parents of the commercial hybrid Suyu 16, which is currently grown in the Jiangsu province of China. XD053 and Y6 differ in multiple traits and are the parents of a recombinant inbred line population developed in our laboratory. To investigate the relationship between RCA genes and grain yield, a total of 123 maize inbred lines from various geographic locations in China or from other origins were chosen for this study. These inbred lines included the parents of the commercial hybrids widely used in China as well as lines derived from Chinese landraces and waxy maize lines. Details of the pedigrees of these inbred lines are provided in Supplemental Table S2. The four inbred lines JB, XD053, Y6, and Y53 were grown under field conditions at the experimental farm of Yangzhou University, Yangzhou, China. Sowing was performed on July 10, 2010. The 123 maize inbred lines were grown under field conditions at the experimental farm of Jiangsu Yanjiang Institute of Agricultural Sciences, Nantong, China. All of the lines were planted in a randomized complete block design with two replications. Each plot included a single row 2.5 m long and 0.60 m wide, with a total of 10 plants and a density of 60,000 plants ha–1. Sowing was performed on March 25, 2011. The cultivation management protocol followed local standard practices at each location.

Tissue Preparation All leaf samples were obtained from the mature upper third of the leaf unless otherwise noted. For each leaf sample, the middle portions of the leaves



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were cut into small pieces and then pooled. At the four-leaf stage, leaf samples were collected from maize plants for cloning and/or western-blot analysis. At the 16-leaf stage, the leaves, roots, stems, immature ears, and tassel spikelets were individually collected for use in the analysis of RCA gene expression levels in different tissues. At 32 DAA, leaf samples obtained at different times (12:30 AM, 4:30 AM, 8:30 AM, 12:30 PM, 4:30 PM) on two successive days were collected to determine RCA gene expression levels during a day/night cycle. At nine developmental stages (four-, eight-, 12-, and 16-leaf and 16, 24, 32, 40, and 46 DAA), leaf samples were collected at approximately 8:30 AM to determine RCA gene expression at the different growth stages. To determine the RCA gene expression levels in the 123 inbred lines, at approximately 30 DAA, the leaves closest to the ear were collected from the plants in each plot in the morning (8:00 AM to 9:30 AM) on a sunny day. All of the samples were collected from three plants of each corresponding inbred line in each plot. After collection, the samples were immediately frozen in liquid nitrogen and subsequently stored at –80°C until further use.

Cloning of the Maize RCA Gene and Sequence Analysis To identify RCA genes in maize, BLASTP searches targeting the maize genome sequence were performed with the Arabidopsis (Arabidopsis thaliana) RCA protein (GenBank accession no. 818558) as the query using the Phytozome Search Tools program (http://www.phytozome.net). The deduced nucleotide sequences of the target genes were downloaded. Consequently, two maize RCA genes were obtained; these were designated ZmRCAa (for the sequence with the longer ORF) and ZmRCAb (for the sequence with the shorter ORF). Based on the sequence information, the genomic DNA and full-length cDNA sequences of ZmRCAa and ZmRCAb were amplified from maize leaves using gene-specific primers (Supplemental Table S1). The amplified products were purified, cloned into the pGEM-T vector (Promega), and subsequently sequenced (Beijing Genomics Institute). Sequence analysis was performed using DNAMAN software (http://www.lynnon.com) and the ChloroP version 1.1 server (http://www.cbs.dtu.dk/services/ChloroP/). The molecular mass of the predicted protein was calculated using the BioXM program (version 2.6; http:// www.bio-soft.net/format/bioxm.htm).

Expression of Recombinant RCA Proteins Using the primers listed in Supplemental Table S1, the ZmRCAa and ZmRCAb cDNAs were used as templates to amplify the truncated ORF lacking a signal sequence. PCR products were digested with BamHI, HindIII, and/or EcoRI restriction enzymes and inserted into the pET-30a expression vector (Novagen). The resulting constructs were introduced into Escherichia coli strain BL21 (DE3; Novagen). The expression of these two recombinant RCA isoforms was performed as previously described (Yin et al., 2010).

Leaf Protein Extraction and Western-Blot Analysis of RCA Protein Leaf protein was extracted using previously described methods (Yin et al., 2010). Protein extracts were subjected to SDS-PAGE using a 12.5% (w/v) acrylamide resolving gel. The separated proteins were then transferred onto polyvinylidene difluoride membranes, and nonspecific antibody binding was blocked with 5% (w/v) nonfat dried milk in phosphate-buffered saline (PBS; pH 7.4) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with polyclonal cotton anti-RCA antibodies (AS10700, Agrisera) diluted 1:10,000 in PBS plus 1% (w/v) nonfat milk. Immune complexes were detected using goat anti-rabbit IgG-horseradish peroxidase (HRP; sc-2004; Santa Cruz Biotechnology). Color was developed with a solution containing 3,39-diaminobenzidine tetrahydrochloride as the peroxidase substrate, and the membranes were scanned.

Relative Quantification of Transcript Expression We used semiquantitative RT-PCR and/or real-time quantitative RT-PCR assays to determine the transcript abundance of ZmRCAa, ZmRCAb, NADP-ME, PPDK, and PEPC. The constitutively expressed Actin gene (GenBank accession no. J01238) was used as an endogenous reference. For the semiquantitative RTPCR assays, 178- and 153-bp fragments of the ZmRCAa and ZmRCAb cDNAs, 2104

respectively, were amplified using Pfu DNA polymerase (Promega). Real-time quantitative RT-PCR was performed according to previously described procedures (Yin et al., 2010). A mixture of cDNA from different inbred lines was used to calibrate each RT-PCR plate. The normalized expression for each line was calculated as delta-delta cycle threshold (DDCT) = (CT, Target – CT, Actin)genotype – (CT, Target – CT, Actin)calibrator. The gene-specific primers used for semiquantitative RT-PCR and real-time quantitative RT-PCR are listed in Supplemental Table S1.

Peptide Synthesis and Polyclonal Antibody Generation Two antigen peptides, N-F-D-P-T-A-R-S-D-D-G-S and A-K-E-V-D-E-T-KQ-T-D, corresponding to the carboxy terminus of predicted ZmRCAa and the amino terminus of predicted ZmRCAb, respectively, were synthesized, and polyclonal antibodies against these peptides were generated in male New Zealand rabbits (GL Biochem). The antibodies were affinity purified on protein A-Sepharose, and their specificity for the corresponding peptides was determined by ELISA. The antibody titers were 1:512,000 and 1:128,000 for the antibodies corresponding to ZmRCAa and ZmRCAb, respectively.

Purification of RCA Polypeptides from Leaves and Recombinant RCA from E. coli After induction with isopropyl-1-thio-b-D -galactopyranoside, E. coli containing recombinant RCA were harvested by centrifugation, resuspended in 8 mL of buffer (50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) containing 2 mM phenylmethylsulfonyl fluoride and disrupted using a sonicator (Tianmei) at 40 W for 3-s bursts on ice for a total time of 10 min. The supernatants obtained after centrifugation (12,000g, 30 min, 4°C) were used for His-tag affinity purification of the recombinant protein. The purification was performed using a Ni2+ column (GenScript) according to the manufacturer’s protocol. Antibodies against ZmRCAa or ZmRCAb (15 mg) were immobilized on 2.5 mL of CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. This resin was used to pour a 1- 3 5-cm column. Pulverized leaf samples were homogenized with extraction buffer and centrifuged for 20 min at 20,000g. Proteins in the supernatant were precipitated with ammonium sulfate at 35% (w/v) saturation, and the precipitate was collected. The resulting pellet was then dissolved in 13 PBS and loaded onto an immunoaffinity column equilibrated with PBS buffer. After washing with 5 column volumes of PBS, the bound RCA was eluted with 50 mM GlyHCl (pH 2.5). Fractions containing RCA were pooled, neutralized by dropwise addition of 250 mM Na2HPO4 (pH 10.3), and stored at –80°C.

Quantification of RCA Polypeptide Expression in Maize Leaves The protein expression levels of ZmRCAa and ZmRCAb were determined on the basis of the total soluble protein content. The total soluble protein concentration of each leaf protein extract was determined using the Bradford assay, and bovine serum albumin (BSA) was used as a standard. The concentration of ZmRCAa or ZmRCAb in each leaf extract sample was measured using an ELISA quantitative sandwich technique modified from Leitao et al. (2003). Nunc Immuno Plates were coated with 100 mL of specific ZmRCAa or ZmRCAb antibodies (1 g mL–1 in 0.1 M Na2CO3/NaHCO3 buffer, pH 9.5). The plates were incubated for 1 h at 37°C and subsequently washed once with PBS-Tween (0.1% [v/v] Tween 20 diluted in PBS [103, Interchim] single strength). To ensure specific fixation of ZmRCAa or ZmRCAb, each well was blocked with 100 mL of 1% (w/v) BSA in PBS-Tween. The plates were incubated for 30 min at 37°C and washed three times with PBS-Tween. Triplicate 100-mL aliquots of each leaf protein extract (each of which had been diluted or concentrated to contain 10 mg of total soluble protein) were dispensed into the wells. After a 1-h incubation at 37°C followed by five washes with PBS-Tween, 100 mL of polyclonal cotton anti-RCA antibodies (5 mg mL–1 in PBS-Tween containing 1% [w/v] BSA) was added to each well. The plates were then incubated for 1 h at 37°C. After five additional washes with PBSTween, immune complexes were detected using goat anti-rabbit IgG-HRP. For color development, 200 mL of a solution containing 3,39,5,59-tetramethylbenzidine liquid substrate (Sigma-Aldrich) was dispensed into each well. The plates were then placed in the dark at room temperature for 30 min. The Plant Physiol. Vol. 164, 2014


Rubisco Activase Genes in Maize reaction was stopped by the addition of 100 mL HCl (1 mol L–1). Lastly, the absorbance was read at 405 nm using a Titertek microplate reader.

MALDI-TOF-MS Analysis of RCA Polypeptides An AB 4800 MALDI-TOF Analyzer (Applied Biosystems, MDS SCIEX) equipped with a 200-Hz neodymium-doped yttrium aluminum garnet laser (355 nm) was utilized to determine the molecular masses of RCA polypeptides. ZipTip C4 was used for the desalting and purification of RCA polypeptide extracts. One microliter of RCA polypeptide extract was spotted onto the MALDI target plate and allowed to air dry. Then, 0.6 mL sinapinic acid matrix was added to the same position on the plate. After the target plate was air dried, MS spectra (20,000–100,000 mass-to-charge ratio [m/z]) were acquired in positive linear ion mode with 7,000 laser shots intensity (25 shots per subspectrum for 500 total shots per spectrum). Mass calibration of MALDI-TOF was achieved using a standard mixture containing aldolase (39,212 m/z) and BSA (66,430 m/z). The parameters for mass assignments were set as follows: minimum signal-to-noise ratio = 20, mass tolerance = 200 m/z, minimum peaks to match = 2, and maximum outlier error = 10 ppm. The 4000 Series Explorer software (V3.5.2) and the Data Explorer software (V4.9; both from Applied Biosystems/MDS SCIEX) were used to perform the MS, data acquisition, and processing.

Grain Yield Measurement Grain yield was estimated using the average yield of five plants in the middle of each row. At maturity, the ears of the corresponding plants were hand harvested, dried to a constant weight, and threshed, and the mean grain yield per plant was recorded.

Statistical Analysis Transcript abundance data for ZmRCAa, ZmRCAb, NADP-ME, PPDK, and PEPC, protein expression data for ZmRCAa and ZmRCAb, and grain yield data for the inbred lines were analyzed using the SAS system (9.0 for Windows). ANOVA was performed using SAS PROC GLM. The mean values of each trait for each inbred line were calculated using SAS PROC MEANS. The Pearson phenotypic correlations among the traits were calculated using SAS PROC CORR. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number JX863889.

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Alternative splicing of ZmRCAb. Supplemental Figure S2. Alignment of ZmRCAa ORFs from four inbred lines. Supplemental Figure S3. MALDI-TOF-MS spectrum of the purified recombinant and leaf RCA proteins. Supplemental Figure S4. Transcript expression levels of ZmRCAa and ZmRCAb determined by semiquantitative RT-PCR. Supplemental Table S1. Primer pairs used in this study. Supplemental Table S2. List of 123 inbred lines and their mean values for different traits. Supplemental Table S3. Descriptive statistics and variance analysis of different traits in 123 lines. Supplemental Table S4. Marker loci associated with ZmRCAa and ZmRCAb transcript abundance and grain yield in 123 lines.

ACKNOWLEDGMENTS We thank Dr. Yuyang Wang of the Testing Center of Yangzhou University for technical assistance with experiments and anonymous reviewers for valuable comments and discussions.

Received October 20, 2013; accepted February 5, 2014; published February 7, 2014.

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