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. 2015 May 5;168(3):1140–1151. doi: 10.1104/pp.15.00580

WHIRLY1 Functions in the Control of Responses to Nitrogen Deficiency But Not Aphid Infestation in Barley1,[OPEN]

Gloria Comadira 1,2, Brwa Rasool 1,2, Barbara Kaprinska 1,2, Belén Márquez García 1,2,2, Jennifer Morris 1,2, Susan R Verrall 1,2, Micha Bayer 1,2, Peter E Hedley 1,2, Robert D Hancock 1,2, Christine H Foyer 1,2,*
PMCID: PMC4741337  PMID: 25944826

A protein that has a role in the control of photosynthetic gene expression and protection against low nitrogen-induced stress.

Abstract

WHIRLY1 is largely targeted to plastids, where it is a major constituent of the nucleoids. To explore WHIRLY1 functions in barley (Hordeum vulgare), RNA interference-knockdown lines (W1-1, W1-7, and W1-9) that have very low levels of HvWHIRLY1 transcripts were characterized in plants grown under optimal and stress conditions. The WHIRLY1-1 (W1-1), W1-7, and W1-9 plants were phenotypically similar to the wild type but produced fewer tillers and seeds. Photosynthesis rates were similar in all lines, but W1-1, W1-7, and W1-9 leaves had significantly more chlorophyll and less sucrose than the wild type. Transcripts encoding specific subsets of chloroplast-localized proteins, such as ribosomal proteins, subunits of the RNA polymerase, and thylakoid nicotinamide adenine dinucleotide (reduced) and cytochrome b6/f complexes, were much more abundant in the W1-7 leaves than the wild type. Although susceptibility of aphid (Myzus persicae) infestation was similar in all lines, the WHIRLY1-deficient plants showed altered responses to nitrogen deficiency, maintaining higher photosynthetic CO2 assimilation rates than the wild type under limiting nitrogen. Although all lines showed globally similar low nitrogen-dependent changes in transcripts and metabolites, the increased abundance of FAR-RED IMPAIRED RESPONSE1-like transcripts in nitrogen-deficient W1-7 leaves infers that WHIRLY1 has a role in communication between plastid and nuclear genes encoding photosynthetic proteins during abiotic stress.

Plastids contain up to 200 copies of the plastid genome that is associated with nonhistone proteins, and they are arranged into discrete protein-DNA complexes, called nucleoids, which are associated with the inner envelope membrane (Krupinska et al., 2013; Powikrowska et al., 2014). Within these structures, single-stranded DNA (ssDNA) binding proteins are responsible for binding and stabilizing the ssDNA until it is used by DNA polymerase or other proteins involved in DNA recombination and repair. Because ssDNA binding proteins must bind all available ssDNA as it becomes accessible, they are highly abundant and fulfill essential roles in DNA replication, recombination, and repair. However, relatively few ssDNA binding proteins have been characterized in plants.

The WHIRLY family of ssDNA binding proteins has a quaternary structure with a whirligig appearance. Like WHIRLY2 (Tarasenko et al., 2012), the WHIRLY1 protein has a preference for binding ssDNA without sequence specificity. It was first characterized as a binding subunit of the nuclear transcriptional activator PATHOGENESIS-RELATED PROTEIN-10a BINDING FACTOR2 (PBF2) that is involved in plant defense gene expression in Solanum tubersum (Desveaux et al., 2002, 2004). WHIRLY proteins have been considered to function as transcription factors in the nucleus, because PBF2 regulates the expression of PR10a by binding to the single-strand form of the elicitor response element (Desveaux et al., 2005). The DNA binding activity of WHIRLY 1 is induced by pathogen elicitors and salicylic acid (SA). It is required for both expression of SA-regulated genes and associated disease resistance, although this pathway is independent of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEIN1 (Desveaux et al., 2004). The barley (Hordeum vulgare) WHIRLY1 protein binds to the promoter of the senescence-associated gene HvS40, which is induced in natural and stress-induced senescence (Krupinska et al., 2002, 2014a).

WHIRLY proteins are found in the chloroplasts and mitochondria as well as the nuclei (Krause et al., 2005; Grabowski et al., 2008). Most plant species, such as barley, have two WHIRLY genes (Desveaux et al., 2005), but there are three WHIRLY genes in Arabidopsis (Arabidopsis thaliana). AtWHIRLY1 and AtWHIRLY3 are targeted to plastids, whereas AtWHIRLY2 is targeted to mitochondria (Krause et al., 2005; Krause and Krupinska, 2009). WHIRLY1 is mainly targeted to plastids, where it is one of the major DNA binding proteins and a major constituent of the plastid nucleoids. In maize (Zea mays), knockout of WHIRLY1 (Zmwhy1-1) by transposon insertion had dramatic effects on plastid gene expression, resulting in albino plants lacking plastid ribosomes with altered splicing of specific transcripts (Prikryl et al., 2008). However, both knockout and knockdown maize lines had equivalent amounts of chloroplast DNA (cpDNA), suggesting that, in maize, WHIRLY1 is not required for cpDNA replication (Prikryl et al., 2008). In contrast, the why1 Arabidopsis mutants have no apparent phenotype (Yoo et al., 2007), except that the seeds showed less sensitivity toward SA and abscisic acid (ABA) than the wild type during germination (Isemer et al., 2012a). When the WHIRLY1 protein was expressed without a targeting sequence (and targeted to the nucleus) of the why1 mutants, the seeds were insensitive to ABA. In contrast, when the wild-type WHIRLY1 protein was targeted to both plastids and the nucleus, the seeds showed enhanced ABA sensitivity (Isemer et al., 2012a).

The double-knockout mutants lacking both WHY1 and WHY3 (why1why3) are largely phenotypically similar to the wild type (Maréchal et al., 2009; Cappadocia et al., 2010). In contrast, a why1why3polIb-1 mutant that is defective in WHY1 and WHY3 as well as DNA polymerase 1B (Pol1B), one of two type I chloroplast DNA polymerases, exhibited a more extreme yellow-variegated phenotype (Lepage et al., 2013). The why1why3polIb-1 mutants had a higher level of illegitimate recombination between repeated sequences and greater plastid genome instability than the wild type (Lepage et al., 2013). Moreover, the why1why3polIb-1 mutants showed lower photosynthetic electron transport efficiencies than the wild-type leaves, and they had a higher accumulation of reactive oxygen species (Lepage et al., 2013). The higher level of oxidation observed in the why1why3polIb-1 triple mutants was linked to chloroplast to nucleus signaling and enhanced adaptation to oxidative stress (Lepage et al., 2013).

The WHIRLY1 protein localizes to the stroma and the thylakoid membranes in maize and barley chloroplasts (Prikryl et al., 2008; Melonek et al., 2010). Although early studies indicated that WHIRLY1 did not associate with plastid nucleoids (Melonek et al., 2010), loss of the WHIRLY1 protein resulted in a slight increase in cpDNA copy number, correlating with increased expression of an organellar DNA polymerase (Krupinska et al., 2014b). Recent evidence from studies using a recombinant form of WHIRLY1 suggested that the protein could translocate from transplastomic chloroplasts to the nucleus (Isemer et al., 2012b). Moreover, WHIRLY1 is a thioredoxin target and hence, a potential target for redox regulation (Foyer et al., 2014).

Current crop yields are highly dependent on significant levels of inorganic nitrogen fertilization, such that chemical nitrogen fixation for fertilizer production is now the largest fraction of total global nitrogen reduction, surpassing both natural biotic and abiotic nitrogen fixation. Thus, a broad range of economic and environmental benefits would follow if crop plants could be selected or engineered to have increased nitrogen use efficiency. A better understanding of the inherent adaptive responses of plants to low-nitrogen availability is, therefore, required to improve nitrogen use efficiency and maintain high productivity on reduced nitrogen inputs. In the following studies, therefore, we have explored the functions of WHIRLY1 in chloroplasts using transgenic barley lines (WHIRLY1-1 [W1-1], W1-7, and W1-9) that have only about 5% or less of the wild-type WHIRLY1 protein (Melonek et al., 2010; Krupinska et al., 2014b) under different growth nitrogen regimes. We present data showing that the WHIRLY1 protein influences the expression of specific subsets of transcripts encoding chloroplast proteins, resulting in a reduced sensitivity of photosynthesis to low nitrogen.

RESULTS

The Phenotype of WHIRLY1-Deficient Barley Seedlings

In these studies, barley seedlings that had been grown in vermiculite with nutrient solution were harvested at time points up to 27 d after germination, which is indicated in the figures. The W1-1, W1-7, and W1-9 barley seedlings had a similar phenotype to the wild type (Fig. 1A), although they had significantly lower levels of WHIRLY1 transcripts (Fig. 1B). In contrast to WHIRLY1, the abundance of WHIRLY2 transcripts was similar in all lines (Fig. 1B). The W1-1, W1-7, and W1-9 barley seedlings accumulated similar amounts of leaf and root biomass (Fig. 1C) to the wild type, with comparable shoot to root ratios (Fig. 1D). Photosynthetic CO2 assimilation rates (Fig. 2A) were similar in the wild type and the W1-1, W1-7, and W1-9 seedlings along with stomatal conductance (Fig. 2B), transpiration rates (Fig. 2C), and partial pressure of CO2 inside the leaf values (Fig. 2D) 4 weeks after germination. However, the W1-1, W1-7, and W1-9 leaves had significantly more chlorophyll and carotenoid pigments than the wild type, with the exception of W1-9 carotenoid content, which was similar to the wild type (Fig. 2E).

Figure 1.

Figure 1.

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Phenotypic comparison of wild-type (WT) seedlings and three independent WHIRLY1-deficient barley lines (W1-1, W1-7, and W1-9). A, Representative appearance of lines 22 d after germination. B, Relative transcript abundance of transcripts encoding Whirly1 and Whirly2 in leaves. C, Shoot and root biomass expressed as fresh weight. D, Shoot to root ratios. Bars in B to D represent means ± se (n = 3). FW, Fresh weight.

Figure 2.

Figure 2.

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Comparisons of photosynthetic gas exchange parameters and leaf pigment contents in wild-type (WT) seedlings and three independent WHIRLY1-deficient barley lines (W1-1, W1-7, and W1-9) measured at 22 or 27 d after germination. Photosynthetic gas exchange parameters (A–d) are shown for individual lines relative to wild-type controls. Mean ± se values (n = 4). Leaf pigment contents (E) estimated at 22 d (n = 4). Car, Carotenoids; Chl, chlorophyll; FW, fresh weight; *, significant difference between the wild type and W1-7 determined by the Student’s t test (P < 0.05).

Line W1-7 has no detectable WHIRLY1 protein and has already been characterized in terms of cpDNA content and nucleoid structure (Krupinska et al., 2014b). We, therefore, performed a more in-depth analysis on the physiology, transcriptome, and metabolome profiles of line W1-7 at the early stages of leaf development. Photosynthesis rates were similar in wild-type and W1-7 seedlings 8 to 14 d after germination (Fig. 3A). However, in very young WHIRLY1-deficient seedlings (i.e. 7 d after germination and younger), photosynthesis rates were lower in W1-7 leaves than the wild type, which was shown by the maximal rates of CO2 assimilation under high light (Fig. 3B). In contrast, at 14 d, the light response curves for photosynthesis were similar in WHIRLY1-deficient and wild-type leaves (Fig. 3, A and C). Stomatal conductance and transpiration rates were higher in the W1 seedlings than in the wild type up to 14 d after germination (Fig. 3, D and E). The total nitrogen content of the shoots and roots was comparable in W1-7 and wild-type seedlings (Supplemental Fig. S1A).

Figure 3.

Figure 3.

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Comparisons of photosynthetic gas exchange parameters in wild-type (WT) and W1-7 seedlings. Photosynthetic CO2 assimilation rates (A), stomatal conductance (D), and transpiration rates (E) measured under 300 mmol m−2 s−1 irradiance from day 8 after germination; data are mean values ± se (n = 3). Light response curves for photosynthesis measured at days 7 (B) and 14 (C) represented as mean ± se values (n = 6). PAR, Photosynthetically active radiation; *, significant difference between the wild type and W1-7 determined by the Student’s t test (P < 0.05).

The WHIRLY1-deficient lines were visibly similar to the wild-type plants throughout development (Supplemental Fig. S2). Seed yields were significantly lower in the WHIRLY1-deficient lines than the wild type, with W1-7 plants exhibiting fewer fertile tillers (Table I). However, seed yield per fertile tiller was similar in W1-7 and wild-type plants (Table I).

Table I. Seed yield in W1-7 and wild-type barley plants.

The numbers of fertile tillers were counted, and total seed yield was quantified in plants grown to maturity in controlled environment greenhouses. Data are presented as the mean values ± se (n = 5). *, P < 0.05 (values that were significantly different between wild-type and W1-7 plants determined using the Student’s t test).

Parameter The Wild Type W1-7
No. of fertile tillers 10.80 ± 1.02* 4.80 ± 1.15*
Total seed yield (g) 13.96 ± 1.68* 5.86 ± 1.36*
Seed yield per fertile tiller (g) 1.28 ± 0.05 0.97 ± 0.24
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The levels of ascorbate, pyridine nucleotides, and glutathione were similar in line W1-7 and wild-type seedlings (Fig. 4). The metabolite profile of the W1-7 leaves was very similar to that of the wild-type seedlings (Fig. 5), indicating a significant difference only in the amount of leaf Suc and a trend toward a lower abundance of reducing sugars and tricarboxylic acid cycle intermediates but no differences in the leaf amino acid pools.

Figure 4.

Figure 4.

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Comparisons of leaf low-molecular weight antioxidant and pyridine nucleotide contents in wild-type (WT) and W1-7 leaves. Total and reduced ascorbic acid (A), reduced and oxidized pyridine nucleotides (B), and total and reduced glutathione were measured on 22-d-old seedlings (C). Data are presented as means ± se (n = 4). AsA, Ascorbic acid; FW, fresh weight; GSH, glutathione.

Figure 5.

Figure 5.

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A comparison of the leaf metabolite profiles of 17-d-old wild-type (WT) seedlings and the WHIRLY1-deficient barley line W1-7 shown as a schematic of key metabolic pathways. Relative metabolite contents were estimated by GC/MS. The bar charts represent the relative concentrations of each metabolite in wild-type (left bars) and W1-7 (right bars) leaves. Data are means ± se (n = 4). GABA, γ-Aminobutyric acid; α-KG, α-ketoglutarate; OAA, oxaloactetate; PEP, phosphoenolpyruvate; 3PGA, 3-phosphoglycerate; *, significant difference between lines estimated using the Student’s t test (P < 0.05).

The Transcript Profile Is Modified in WHIRLY1-Deficient Leaves

The transcript profile of the W1-7 leaves was characterized by increases in the abundance of large numbers of transcripts encoding proteins involved in photosynthesis and protein synthesis (Fig. 6A; Supplemental Table S1). The differences in transcript abundance between W1-7 and wild-type leaves were confirmed by quantitative PCR (Fig. 6B).

Figure 6.

Figure 6.

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Transcript profile comparison of 14-d-old wild-type leaves and the WHIRLY1-deficient barley W1-7 line. Transcripts that were significantly differentially abundant in W1-7 leaves relative to the wild type were categorized according to the function of encoded proteins (A). Microarray quantitative reverse transcription (qRT)-PCR comparisons of selected transcripts indicated the validity of the array data (B). The relative abundance of transcripts encoding proteins of known function is indicated (C) according to the functional classification described in A and detailed in Supplemental Table S1. Genes were identified using a moderated Students t test with Benjamini-Hochberg multiple testing correction (P < 0.05; Fold change > 2; Genespring 12; Aligent Technologies).

Transcripts that were much more abundant in W1-7 leaves include a component of the DNA polymerase type 1 complex (Morex Locus [MLOC] 54735.1) that functions in the replication and repair of plastid DNA. This transcript is identical to the barley organelle DNA polymerase described recently (Krupinska et al., 2014a, 2014b) and homologous to the AtPol1B (At3g20540) sequence. Similarly, a microtubule end binding protein 1A (MLOC 52339.1) and a transcript encoding a protein containing a B3 DNA binding domain (AK251585.1) that is found exclusively in transcription factors were significantly more abundant in W1-7 leaves relative to the wild type (Fig. 6C; Supplemental Table S1). The transcript profile of the W1-7 leaves showed no changes in the innate immune responses of the plants. To confirm the absence of effects on the innate immune responses, we compared the susceptibility of the W1-1, W1-7, and W1-9 lines with aphid (Myzus persicae) infestation and found that aphid fecundity was similar in the WHIRLY1-deficient and wild-type lines (Supplemental Fig. S3).

A large number of transcripts encoding chloroplast-associated proteins was significantly increased in abundance in W1-7 leaves relative to the wild type (Table II). These include a number of components associated with the thylakoid NADH dehydrogenase complex (NDHA, NDHC, NDHD, NDHF, NDHB.2, NDHH, NDHJ, NDHI, and NDHG), the chloroplast RNA polymerase (RPOC2, RPOB, and RPOC1), the cytochrome b/f complex (PHOTOSYNTHETIC ELECTRON TRANSFER [PET]A, PETD, and a transcript encoding the cytochrome C biogenesis protein YCF5), chloroplast ribosomes (RPL20, RPL23.2, RPL33, and RPS2), and minor components associated with the PSII (PSBF, PSBB, and PSBJ) and PSI (PSAC and PSAJ) reaction centers (Table II).

Table II. Barley transcripts homologous to plastid-encoded genes in Arabidopsis that exhibit significant differences in abundance in wild-type and W1-7 seedlings (except for those marked with an asterisk, which are nuclear encoded).

Accession No.a Transcript Abundance W1-7/the Wild Typeb Top Arabidopsis Matchc Arabidopsis Gene Descriptiond
MLOC 24854.1 8.59 AtCg01050 NDHD, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 24746.1 4.76 AtCg00190 RPOB, RNA polymerase subunit-β*
MLOC 1704.1 4.74 AtCg00190 RPOB, RNA polymerase subunit-β*
MLOC 54708.1 4.56 AtCg00170 RPOC2, RNA polymerase family protein*
MLOC 24776.1 3.96 AtCg01040 YCF5, Cyt C assembly protein*
MLOC 9149.1 3.24 AtCg01010 NDHF, NADH-ubiquinone oxidoreductase (complex I), chain 5 protein
MLOC 9313.1 3.09 AtCg00040 MATK, maturase K
MLOC 25280.1 3.047 AtCg01010 NDHF, NADH-ubiquinone oxidoreductase (complex I), chain 5 protein
MLOC 61567.1 2.92 AtCg01010 NDHF, NADH-ubiquinone oxidoreductase (complex I), chain 5 protein
MLOC 456.1 2.87 AtCg00170 RPOC2, RNA polymerase family protein*
MLOC 32552.1 2.87 AtCg01300 RPL23.2, ribosomal protein L23
MLOC 34251.1 2.81 AtCg01050 NDHD, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 24733.1 2.81 AtCg00180 RPOC1, RNA polymerase family protein*
MLOC 24753.1 2.78 AtCg01050 NDHD, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC24802.1 2.63 AtCg00040 MATK, maturase K
MLOC 36249.1 2.62 AtCg01050 NDHD, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 26369.1 2.61 AtCg01250 NDHB.2, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 63387.1 2.58 AtCg01110 NDHH, NAD(P)H dehydrogenase subunit H
MLOC 9538.1 2.53 AtCg00180 RPOC1, RNA polymerase family protein*
MLOC 34273.1 2.47 AtCg00040 MATK, maturase K
MLOC 8394.1 2.47 AtCg00360 YCF3, tetratricopeptide repeat-like superfamily protein
MLOC 33340.1 2.40 AtCg01250 NDHB.2, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 365.2 2.34 AtCg01250 NDHB.2, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 77504.1 2.23 AtCg01250 NDHB.2, NADH-ubiquinone/plastoquinone (complex I) protein
MLOC 6335.1 2.16 AtCg00590 ORF31, electron carriers
MLOC 36200.1 2.11 AtCg00730 PETD, photosynthetic electron transfer D
MLOC 36158.1 2.11 AtCg00590 ORF31, electron carriers
MLOC 9573.1 2.10 AtCg00170 RPOC2, RNA polymerase family protein*
MLOC 2607.1 2.06 AtCg00570 PSBF, PSII reaction center protein F
MLOC 24780.1 2.03 AtCg00730 PETD, photosynthetic electron transfer D
MLOC 24745.1 2.02 AtCg00660 RPL20, ribosomal protein L20
MLOC 9018.1 2.00 AtCg00640 RPL33, ribosomal protein L33
MLOC 8945.1 1.95 AtCg00420 NDHJ, NADH dehydrogenase subunit J
MLOC 7873.1 1.95 AtCg01090 NDHI, subunit of the chloroplast NAD(P)H dehydrogenase complex
MLOC 9520.1 1.93 AtCg00470 ATPE, ATP synthase ε-chain
MLOC 3829.1 1.93 AtCg00530 YCF10
MLOC 8957.1 1.91 AtCg00420 NDHJ, NADH dehydrogenase subunit J
MLOC 9641.1 1.90 AtCg00530 YCF10
MLOC 36232.1 1.88 AtCg00160 RPS2, ribosomal protein S2
MLOC 9681.1 1.87 AtCg00530 YCF10
MLOC 61555.1 1.86 AtCg01080 NDHG, NADH-ubiquinone/plastoquinone oxidoreductase, chain 6
MLOC 8426.1 1.85 AtCg00530 YCF10
MLOC 12189.1 1.82 AtCg00420 NDHJ, NADH dehydrogenase subunit J
MLOC 65510.1 1.82 AtCg01090 NDHI, subunit of the chloroplast NAD(P)H dehydrogenase complex
MLOC 24811.1 1.78 AtCg00420 NDHJ, NADH dehydrogenase subunit J
MLOC 41067.1 1.77 AtCg00630 PSAJ, PSI subunit J
MLOC 68023.1 1.76 AtCg00530 YCF10
MLOC 34111.1 1.75 AtCg00540 PETA, photosynthetic electron transfer A
MLOC 9478.1 1.69 AtCg00160 RPS2, ribosomal protein S2
MLOC 61315 1.68 AtCg00480 ATPB, ATP synthase subunit-β
MLOC 9572.1 1.64 AtCg01100 NDHA, NADH dehydrogenase family protein
MLOC 59993.1 1.59 AtCg00440 NDHC, NADH dehydrogenase D3 subunit
MLOC 63096.1 1.31 AtCg00680 PSBB, PSII reaction center protein B
MLOC 9455.1 1.30 AtCg00680 PSBB, PSII reaction center protein B
MLOC 9691.1 1.22 AtCg00550 PSBJ, PSII reaction center protein J
MLOC 24726.1 0.61 AtCg01060 PSAC, PsaC subunit of PSI
MLOC 9445.1 0.45 AtCg01110 NDHH, NAD(P)H dehydrogenase subunit H
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a

Barley gene model primary accession number (Mayer et al., 2012).

b

Transcript abundance in W1-7 seedlings relative to transcript abundance in wild-type seedlings.

c

The Arabidopsis Information Resource accession of top Arabidopsis match based on BLAST e value.

d

The Arabidopsis Information Resource annotation of Arabidopsis gene.

WHIRLY1-Deficient Seedlings Show Enhanced Resistance to Nitrogen Deficiency

The wild-type and W1-1, W1-7 and W1-9 lines showed similar low nitrogen-dependent decreases in shoot (Fig. 7A) and root (Fig. 7B) biomass. The W1-1, W1-7, and W1-9 leaves had 3-fold more chlorophyll than the wild type under nitrogen deficiency (Fig. 7C). In contrast to the wild-type leaves, which were only capable of respiration after 22 d of nitrogen deficiency, the W1-1, W1-7., and W1-9 leaves were still able to undertake photosynthetic CO2 assimilation (Fig. 7D). The carbon contents of the wild-type and W1-7 lines were similar, irrespective of nitrogen supply (Supplemental Fig. S1, B and E), whereas both lines exhibited a similar decrease in nitrogen content and increase in the carbon to nitrogen ratio under nitrogen limitation (Supplemental Fig. S1, A, C, D, and F).

Figure 7.

Figure 7.

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Impact of nitrogen availability on growth and photosynthesis in wild-type (WT) seedlings and three independent WHIRLY1-deficient barley lines (W1-1, W1-7, and W1-9). Plants were grown for 22 d under either optimal (N replete) or low-nitrogen (N deficient) conditions. Shoot (A) and root (B) biomass was estimated after destructive harvesting of plants, and values are represented as means ± se (n = 3). Chlorophyll content (C) was estimated after extraction from harvested shoots, and CO2 assimilation rate (D) was estimated by gas exchange before harvest; values are represented as means ± se (n = 4). FW, Fresh weight; *, significant differences between WHIRLY1-deficient and wild-type plants estimated by the Student’s t test (P < 0.05).

The Transcript Profile of WHIRLY1-Deficient Leaves Exhibits a Distinct Response to Low Nitrogen

WHIRLY1 transcripts were increased in the leaves of the wild-type plants grown with low nitrogen (Fig. 8A). In contrast, the levels of WHIRLY1 transcripts were much lower in W1-1, W1-7, and W1-9 lines than the wild type under both nutrition regimes (Fig. 8A). However, WHIRLY2 transcripts were increased to a similar extent under the low-growth nitrogen regimes in all lines (Fig. 8B). The levels of psbA, psbJ, ndhA, and ndhJ were significantly lower in the W1-1, W1-7, and W1-9 lines than the wild type when expressed relative to the chloroplast 16S ribosomal transcripts under optimal nitrogen (Fig. 8C). However, the levels of these transcripts relative to the chloroplast 16S ribosomal transcripts were similar in all lines under the low-growth nitrogen regime (Fig. 8D).

Figure 8.

Figure 8.

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Abundance of transcripts encoding WHIRLY and several chloroplast-encoded proteins in leaves of the wild type (WT) and three independent WHIRLY1-deficient barley lines (W1-1, W1-7, and W1-9). Plants were grown for 15 d under either optimal (N replete) or low-nitrogen (N deficient) conditions. The abundance of transcripts encoding WHIRLY1 (A) and WHIRLY2 (B) was estimated relative to their abundance in wild-type plants under nitrogen-replete conditions by the ΔΔCT method (Schmittgen and Livak, 2008) using actin as a reference. The abundance of chloroplast-encoded transcripts was estimated relative to their abundance in wild-type plants under nitrogen-replete (C) or -deficient (D) conditions using 16S ribosomal RNA as a reference. All data are presented as mean values ± sd (n = 3).

To analyze the effects of nitrogen regime on the leaf transcript profile, W1-7 and wild-type seedlings were grown for 15 d under either optimal nitrogen conditions or nitrogen deficiency. About 50% of the transcripts that were more abundant in W1-7 leaves under nitrogen-replete conditions were also more abundant in conditions of nitrogen deficiency (Fig. 9A). Another 40 transcripts were induced in W1-7 leaves under conditions of nitrogen deficiency (Fig. 9A). The marked difference between the leaf transcriptome profiles in seedlings grown under nitrogen deficiency and those grown under nitrogen-replete conditions was the high number of transcripts involved in RNA processing and signaling, which was increased in W1-7 leaves relative to the wild type (Fig. 9, C and D). Of note was the large decrease in the abundance of transcripts encoding Eukaryotic Initiation Factor 4A (eIF4A; Supplemental Table S1), a component of the eIF4F complex, which recognizes the 7-methylguanosine cap of mRNA and is involved in the initiation phase of eukaryotic translation (Aitken and Lorsch, 2012). Together with eIF4G, which serves as a scaffold to recruit other translation initiation factors, eIF4F ultimately assembles the 80S ribosome. In addition, the levels of transcripts encoding a FAR-RED IMPAIRED RESPONSE1 (FAR1)-like protein were greatly increased in the W1-7 compared with the wild type under conditions of nitrogen deficiency along with transcripts encoding a putative His kinase and a Leu-rich repeat transmembrane receptor-like kinase of the STRUBBELIG (SUB) family (Supplemental Table S1).

Figure 9.

Figure 9.

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Transcript profile comparison of 15-d-old wild-type leaves and the WHIRLY1-deficient barley W1-7 line grown under either optimal (N replete) or low-nitrogen (N deficient) conditions. A, Venn diagram illustrating the number of differentially abundant transcripts under each nitrogen regime. B, Classification of transcripts that showed differential abundance only under nitrogen-replete conditions. C, Classification of transcripts that showed differential abundance under both nitrogen conditions. D, Classification of transcripts that showed differential abundance only under conditions of nitrogen deficiency. The differentially expressed genes were identified using a moderated Student's t test with Benjamini-Hochberg multiple testing correction (P < 0.05; Fold change > 2; Genespring 12; Aligent Technologies).

DISCUSSION

The WHIRLY1 protein is targeted to plastids, where it is one of the major DNA binding proteins and a constituent of the nucleoids. In these studies, we have characterized WHIRLY1 functions in independent transgenic barley lines (W1-1, W1-7, and W1-9) that have very low levels of HvWHIRLY1 transcripts. The loss of WHIRLY1-dependent controls had little effect on photosynthetic CO2 assimilation rates, except at the early stages of seedling development. However, leaves lacking WHIRLY1 accumulate more chlorophyll after 7 d of seedling development under optimal growth conditions and conditions of nitrogen limitation. The differences between the results presented here and those obtained in chloroplasts isolated from these RNA interference lines (Melonek et al., 2010) may be explained by differences in the growth irradiances used in the two studies. The data presented here show that barley leaves that have low expression of WHIRLY1 accumulate more chlorophyll, even under conditions of nitrogen deficiency.

The role of WHIRLY 1 in expression of SA-regulated genes and pathogen resistance (Desveaux et al., 2004) led us to address the question of whether WHIRLY1-deficient barley seedlings showed altered responses to aphid infestation. However, the data in Supplemental Figure S3 show that aphid fecundity was similar in all lines. This observation is consistent with the absence of changes in transcripts encoding pathogen-related signaling pathways in the W1-7 leaf profile. Moreover, the significantly lower Suc content of the W1-7 leaves relative to the wild type did not affect the ability of aphids to infest the leaves.

The transcript profile analysis of the WHIRLY-deficient leaves showed that genes encoding discrete subsets of photosynthetic proteins were markedly changed relative to the wild type. These findings are consistent with the observation that barley leaves seem to be less able to regulate cpDNA copy number in the absence of WHIRLY1 (Krupinska et al., 2014b). Transcripts encoding photosynthetic proteins, including the thylakoid NADH dehydrogenase and cytochrome b/f complexes, and chloroplast ribosomes were more abundant in the WHIRLY-deficient leaves than the wild type. Plastid DNA contains 11 NADPH dehydrogenase (ndh) genes, which are significantly represented in the W1-7 profile (Table II). The chloroplast NADH complex, which is composed of chloroplast- and nuclear-encoded subunits, functions in cyclic electron flow around PSI (Peng et al., 2009, 2011) and protection against stress (Casano et al., 2001). The data shown in Table II suggest that WHIRLY1 functions to suppress the expression of genes encoding intersystem carriers, such as the cytochrome b/f and NDH complexes, and may, therefore, be important in the regulation of cyclic electron flow (Miyake, 2010; Foyer et al., 2012). Although these changes may be linked to a failure to regulate cpDNA copy number (Krupinska et al., 2014b), it is interesting that the observed increases in these transcripts are not accompanied by similar changes in the expression of nuclear genes encoding photosynthetic proteins. The expression of nuclear genes encoding photosynthetic proteins, therefore, seems to be uncoupled to some extent from plastid gene expression in the WHIRLY-deficient leaves.

The data presented here show that transcripts encoding core PSI and PSII reaction center complexes and light harvesting components were unaffected by WHIRLY1. This finding is surprising given the sensitivity of PSI and PSII gene expression to light and metabolic controls (Foyer et al., 2012). The absence of any effects of WHIRLY1 deficiency on PSI and PSII gene expression may explain why there were few detectable differences in photosynthetic CO2 assimilation rates between the WHIRLY1-deficient and wild-type leaves in the absence of stress. However, the W1-7 leaves had significantly less Suc than the wild type, suggesting that the influence of WHIRLY1 extends to carbon metabolism.

The why1why3polIb-1 mutants of Arabidopsis exhibited higher plastid genome instability and enhanced oxidative stress (Lepage et al., 2013). The data presented here show that transcripts encoding a chloroplast-targeted Cu, Zn superoxide dismutase2 (SOD2) were much more abundant in WHIRLY1-deficient leaves than wild-type controls along with transcripts encoding phytochelatin synthase that functions in xenobiotic metabolism and the catabolism of glutathione conjugates (Fig. 9; Supplemental Table S1). However, the levels of the reduced and oxidized forms of the major leaf antioxidants ascorbic acid and glutathione as well as pyridine nucleotides were not changed in the WHIRLY1-deficient leaves, suggesting that, if WHIRLY1 deficiency leads to enhanced production of reactive oxygen species, it does not cause a global change in the cellular redox state but rather, a localized increase in oxidation (for example, in the plastids). The increase in the levels of transcripts encoding chloroplast-targeted Cu, Zn SOD2, which is a nuclear gene, lends support to the hypothesis that WHIRLY1 is involved in chloroplast to nucleus signaling (Lepage et al., 2013).

The data presented here show that the regulation of plastid DNA replication by WHIRLY1 has wide-reaching implications for other pathways and processes influenced by chloroplast-derived signals. However, transcripts encoding transcription factors and stress signaling proteins were not significantly altered in abundance by WHIRLY1 deficiency. The absence of a changed stress response was confirmed by the similar levels of aphid infestation observed in wild-type and WHIRLY1-deficient leaves. Transcripts encoding His kinases and SUB, which is a receptor-like kinase involved in intercellular signal transduction (Eyüboglu et al., 2007), were increased in WHIRLY1-deficient leaves. A transcript that shows homology to an Oryza sativa plant-specific type II MIKC MADS box gene was increased in abundance in W1-7 leaves relative to the wild type. The transcription factor of this type that has been most intensively studied, the wheat (Triticum aestivum) AGAMOUS-like33 (TaAGL33), is a key regulator of developmental processes, such as meristem identity, flowering time, and fruit and seed development (Winfield et al., 2009). The expression of TaAGL33 represses flowering and cell elongation by down-regulation of a group of genes related to the Arabidopsis FLOWERING PROMOTING FACTOR1 (Greenup et al., 2011). The enhanced abundance of the type II MIKC MADS box transcripts in W1-7 plants may explain, at least in part, why the WHIRLY1-deficient barley plants show altered development with fewer fertile tillers than the wild type (Table I).

The WHIRLY1-deficient barley seedlings were more resistant to nitrogen deficiency in terms of decreased loss of leaf chlorophyll and less inhibition of photosynthetic CO2 assimilation rates, suggesting that the failure to control cpDNA copy number and repress plastid DNA replication exerts an influence on the systems that decrease photosynthesis in response to nitrogen deficiency. Although the data presented here do not give many insights into the mechanisms that underpin this response, it is interesting to note that FAR1-like protein transcripts were much more abundant in the W1-7 leaves than the wild type under conditions of nitrogen deficiency. Although the molecular nature of plastid to nucleus signaling pathways remains poorly understood, the transcriptional control in response to light is closely tied to the primary signaling function of the phytochrome system. The FAR1 gene encodes a transposase-related transcription factor that activates the expressions of FAR-RED ELONGATED HYPOCOTYL1 and FHY1-LIKE, which promote the nuclear translocation of phytochrome A, resulting in the activation of phytochrome A-mediated gene expression, such as chloroplast division and chlorophyll biosynthesis (Tang et al., 2012). The FAR1 protein also acts as a positive regulator of ABA signaling in Arabidopsis, enabling adaptation to environmental stresses (Tang et al., 2013). WHIRLY1 has been shown to influence the responsiveness of seedlings to ABA (Isemer et al., 2012a). The high abundance of transcripts encoding an FAR1-like protein in the leaves of W1-7 grown under low nitrogen may explain the higher chlorophyll levels observed in WHIRLY1-deficient leaves if they are linked to increased chlorophyll synthesis through modulation of HEMB1 as described previously (Tang et al., 2012).

In conclusion, the data presented here show that WHIRLY1 is an important regulator of cpDNA and plastid gene expression. Severely reduced expression of WHIRLY1 leads to a disruption of the communication between the plastid and nuclear genes encoding photosynthetic proteins, leading to an accumulation of chlorophyll in leaves that persists even under nitrogen limitation. Moreover, photosynthesis was less susceptible to inhibition in plants grown under low nitrogen, suggesting that WHIRLY1 is a potentially previously unrecognized target for improvement of nitrogen use efficiency.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of three independent transgenic barley (Hordeum vulgare ‘Golden Promise’) lines (W1-1, W1-7, and W1-9) with RNAi knockdown of the WHIRLY1 gene and wild-type controls were produced as described previously (Melonek et al., 2010; Krupinska et al., 2014b). Seeds were allowed to germinate for 7 d in the absence of added nitrogen. They were then sown in pots in vermiculite in controlled environment chambers with a 16-h-light/8-h-dark photoperiod (irradiance = 450 µmol m−2 s−1), 21°C/16°C day-night temperature regime, and 60% relative humidity. The pots were arranged in trays (16 pots per tray). Every 2 d, each tray was provided with 2 L of the nutrient solution described by Møller et al. (2011) consisting of 0.2 mm KH2PO4, 0.2 mm K2SO4, 0.3 mm MgSO4·7H2O, 0.1 mm NaCl, 0.1 µm MnCl2, 0.8 µm Na2MoO4·2H2O, 0.7 µm ZnCl2, 0.8 µm CuSO4·5H2O, 2 µm H2BO3, 50 µm Fe(III)-EDTA-Na, and either 5 mm KNO3 (nitrogen replete) or 0.1 mm KNO3 (nitrogen deficient). Plants were harvested 7, 9, 17, 22, or 27 d after the initiation of the low-nitrogen and optimal nitrogen treatment regimes.

For analysis of seed yield, plants were grown to maturity in compost in a standard heated greenhouse at 22°C under a 16-h photoperiod, where supplementary lighting was provided by high-pressure sodium vapor lamps (Powertone SON-T AGRO 400W; Philips ElectronicUK).

Shoot and Root Biomass

Whole plants were harvested and separated into shoots and roots. These were weighed immediately and then dried in an oven at 80°C for 2 d, after which the tissues were weighed again.

Photosynthesis Measurements

Photosynthetic gas exchange measurements were performed using a portable system Ciras-2 Infrared Gas Analyzer (model ADC 225 Mark 3; The Analytical Development Co. Ltd.) set at 300 μmol m−2 s−1 of photosynthetically active radiation, 40% to 50% relative humidity in the leaf chamber, and leaf chamber CO2 and O2 concentrations maintained at 400 ± 10 and 210 μmol mol−1, respectively. The temperature of the leaf chambers was set at 20°C ± 0.5°C. Calculations of CO2 assimilation rate and stomatal closure were performed as described previously (Von Caemmerer and Farquhar, 1981).

Aphid Fecundity Assays

Barley plants were grown for 15 d under nitrogen-replete conditions as described above. At the end of this period, a single 1-d-old nymph of aphid (Myzus persicae) was applied to the lamina of the oldest leaf, and plants were individually caged inside clear plastic containers (10-cm i.d. × 20-cm height) and capped with a 200-μm mesh. Plants were provided with nutrient solution weekly and after 15 d, carefully removed from the cages; aphids present were counted under a hand lens.

Leaf Pigment Content

Leaf pigments were extracted and analyzed according to the method by Lichtenthaler (1987).

Carbon and Nitrogen Content

The carbon and nitrogen contents were determined on the dried leaf and root material from five biological replicates per genotype per treatment using an LECO Trumac Combustion Analyzer (Yara UK Limited Company).

Ascorbate, Glutathione, and Pyridine Nucleotide Assays

Ascorbate, glutathione, and pyridine nucleotides were extracted and analyzed as described by Queval and Noctor (2007).

Metabolite Analysis by Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry (GC/MS) analysis was performed on extracts from four biological replicates per genotype per treatment. Leaf samples were lyophilized, and polar and nonpolar extracts were prepared, derivatized, and analyzed by GC/MS (Dobson et al., 2008). Data were then processed using Xcalibur software.

Microarray Processing and Analysis

Microarray processing was performed on leaf RNA extracts from four biological replicates per genotype per treatment using a custom-designed barley Agilent Microarray (A-MEXP-2357; www.ebi.ac.uk/arrayexpress). The microarray contains approximately 61,000 60-mer probes derived from predicted barley transcripts and full-length complementary DNAs (Mayer et al., 2012).

These probes were selected from a total of approximately 80,000 predicted genes by prioritizing them according to their annotation (section S7.1.4 and supplemental fig. S18 in Mayer et al., 2012). The high-confidence gene set was used in its entirety (n = 26,159). This set of predicted genes is based on being supported by homology to at least one closely related species (Brachypodium distachyon, Sorghum bicolor, Oryza sativa, and Arabidopsis [Arabidopsis thaliana]). Next, 14,481 genes were added that had been annotated as remote homologs based on a lack of homology to monocot proteins. We also added 7,999 genes from the Triticeae-specific category, which is defined as having significant BLASTN hits to the wheat (Triticum aestivum) full-length complementary DNA library but no significant BLASTX hit to angiosperm reference protein sequences. The remainder of sequences on the chip (n = 12,848) derive from genes that had no homology to any of the databases used and are assumed to be specific to barley. This resulted in a total of 61,487 genes represented on the chip by a single probe sequence each.

Microarray processing was performed according to the One-Color Microarray-Based Gene Expression Analysis protocol (version 6.5; Agilent Technologies). Data were extracted using Feature Extraction software (version 10.7.3.1; Agilent Technologies) with default settings and subsequently analyzed using GeneSpring GX (versions 7.3 and 12; Agilent Technologies) software. Data were normalized using default Agilent Feature Extraction one-color settings in GeneSpring and filtered to remove inconsistent probe data flagged as absent in more than one replicate per sample. Probes were identified as significantly changing between genotype and nitrogen treatment using two-way ANOVA with a P value of <0.05 with Bonferroni multiple testing correction. Probes were identified as significantly changing between WHIRLY and the wild type under sufficient or deficient nitrogen treatments by a moderate Students t test and Benjamini-Hochberg false discovery rate multiple testing correction (P < 0.05; fold change > 2).

Raw data from this article can be found at the Array Express Web site (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2242.

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Comparisons of shoot and root carbon and nitrogen contents in wild-type and WHIRLY1-deficient W1-7 seedlings.

  • Supplemental Figure S2. Appearance of wild-type and WHIRLY1-deficient barley lines grown under optimal nitrogen.

  • Supplemental Figure S3. Aphid fecundity on wild-type and WHIRLY1-deficient barley lines.

  • Supplemental Table S1. Transcripts with significantly different abundance in W1-7 and wild-type barley leaves under differing nitrogen availability.

Acknowledgments

We thank Karin Krupinska for providing the inspiration and motivation for these studies and supplying the seeds of the transgenic barley lines used in the experiments and Dr. Jennifer Stephens for performing the seed yield measurements.

Glossary

ABA

abscisic acid

GC/MS

gas chromatography-mass spectrometry

SA

salicylic acid

ssDNA

single-stranded DNA

Footnotes

1

This work was supported by the European Union Marie-Curie Initial Training Network (Initial Training Network Croplife Project no. PITN–GA–2010–264394), the Biological Sciences Research Council, UK (grant no. BB/M009130/1), and the Rural and Environment Science and Analytical Services Division of the Scottish Government (to J.M., S.R.V., M.B., P.E.H., and R.D.H.).

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