Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis.

Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues.


INTRODUCTION
Chloroplasts are organelles that convert light energy into chemical energy to sustain plant growth. Chloroplasts not only carry out photosynthesis but also play a pivotal role in plant metabolism, being involved in the biosynthesis of amino acids, fatty acids, and phytohormones (Neuhaus and Emes, 2000). Chloroplasts of vascular plants develop from a non-photosynthetic progenitor, a proplastid, which is maintained in the meristematic cells.
Proplastids are colorless and contain limited amounts of internal membranes, but they can differentiate into a variety of plastid types with specialized activities, such as amyloplasts in the roots for starch storage, leucoplasts for lipid storage, chromoplasts for pigment accumulation, etioplasts in dark-grown shoots, and chloroplasts in light-grown shoots. The different types of plastids are interconvertible in response to developmental and environmental changes (Mullet, 1988;López-Juez and Pyke, 2005). In the absence of light, proplastids can differentiate into etioplasts which contain a semi-crystalline structure called the prolamellar body with precursors of both chlorophyll and thylakoid membrane lipids (Armstrong et al., 1995). Upon illumination, genes associated with biogenesis of the mature chloroplast are expressed, and several subsequent activities, including import of nuclear-encoded chloroplast proteins, expression of plastid-encoded photosynthetic genes, biosynthesis of chlorophyll, assembly of the photosystems and the thylakoid network, and chloroplast division, occur in parallel to complete chloroplast development.
Multiple factors regulate chloroplast development, light being chief among these factors. Phytohormones, in particular cytokinins, can also modulate chloroplast development. For example, chloroplast maturation is induced by cytokinins in tobacco tissue cultures (Stetler and Laetsch, 1965); exogenous cytokinins induce re-greening of senescent Nicotiana leaves ; and cytokinins activate chloroplast differentiation from proplastids in pumpkin and watermelon cotyledons (Khokhlova, 1977;Longo et al., 1979). In addition, transcription of several photosynthetic genes encoded in the plastids of barley is increased by exogenously applied cytokinins (Zubo et al., 2008). Similarly, seedling de-etiolation is induced by cytokinins (Chory et al., 1994) and many proteins related to chloroplast biogenesis are up-regulated in response to cytokinins in Arabidopsis (Lochmanová et al., 2008). In spite of the significance of cytokinins for chloroplast biogenesis, the molecular mechanism of their actions on chloroplasts is largely unknown.
Chloroplast biogenesis requires the coordinated transcriptional regulation of genes encoded in the nuclear and chloroplast genomes. Thus, one might predict the existence of nuclear transcription factors that regulate the expression of genes required for chloroplast biogenesis. However, very few transcription factors functioning as positive regulators of chloroplast biogenesis have been identified. A notable exception is the GOLDEN TWO-LIKE (GLK) family of transcription factors (Fitter et al., 2002;Waters et al., 2009). The glk1 glk2 double mutant is pale green with a severe reduction in thylakoids, and the GLK transcription factors promote the expression of many nuclear-encoded photosynthetic genes that are associated with chlorophyll biosynthesis and light harvesting functions (Waters et al., 2009). In addition, and tying back to the role of cytokinin in regulating chloroplast biogenesis, overexpression of the cytokinin-regulated transcription factor chloroplast division rate by up-regulating the protein level of PLASTID DIVISION 2 (PDV2) (Okazaki et al., 2009).

GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and
CYTOKININ-RESPONSIVE GATA1 (CGA1) were first identified based on their induction by nitrate (Wang et al., 2003;Price et al., 2004;Scheible et al., 2004;Bi et al., 2005). More recent studies indicate that expression of this family is also induced by light and cytokinin (Naito et al., 2007), and is repressed during flower development (Mara and Irish, 2008). Functionally, they have been shown to antagonize signaling by gibberellins, acting downstream of DELLA proteins (Richter et al., 2010). Effects on greening have also been observed in genetic studies, with the gnc cga1 mutant exhibiting reduced greening compared to wild-type and overexpression of either gene resulting in enhanced greening and chloroplast production (Bi et al., 2005;Naito et al., 2007;Mara and Irish, 2008;Richter et al., 2010;Hudson et al., 2011;Köllmer et al., 2011). The chloroplast-localized gene GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), a key gene involved in nitrogen assimilation, has been identified as a potential transcriptional target of GNC and CGA1 (Hudson et al., 2011). However, thus far a detailed picture of the physiological function of this GATA family of transcriptional regulators is lacking. We hypothesize that these transcription factors are part of a pathway that links light and cytokinin signaling with the differentiation of photosynthetically active chloroplasts from proplastids and their proliferation throughout plant development. To characterize these two proteins functionally and to place them into the pathway that mediates chloroplast development, we performed an extensive physiological analysis of both loss-and gain-of-function lines. This approach hypocotyl. In addition, some weaker staining is found in the primary roots. In 14-day-old seedlings, staining for both GNC and CGA1 became more pronounced in the green shoot tissues, consistent with the strong expression identified in shoots by qRT-PCR (Fig. 1A).
To determine the subcellular localization of GNC and CGA1, the genomic sequence of these genes was translationally fused to GFP and transiently expressed from the CaMV 35S promoter in Arabidopsis mesophyll protoplasts. In agreement with their roles as transcription factors, both GNC and CGA1 co-localized with a histone-2B-RFP fusion protein in the nucleus (Supplemental Fig. S2).

GNC and CGA1 Regulate Chlorophyll Production
To characterize the function of GNC and CGA1, T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center. Both gnc (SALK_001778) and cga1 (SALK_003995) homozygous lines resulted in undetectable levels of full-length transcripts (Supplemental Fig. S3B). In agreement with previous reports that gnc and cga1 mutants have defects in greening (Bi et al., 2005;Mara and Irish, 2008;Richter et al., 2010), the gnc single mutant and gnc cga1 double mutant displayed significantly reduced chlorophyll levels in the shoots (Supplemental Fig. S4). The pale green phenotype of the gnc single mutant and the higher basal levels of GNC transcripts compared to CGA1 transcripts (Naito et al., 2007) suggest that GNC plays the more dominant role in regulating chlorophyll production.
The decreased chlorophyll levels in the gnc cga1 mutant suggest a role of this gene family in a chloroplast function. However, this pale-green phenotype alone does not indicate a normal role in the control of chloroplast development, because loss of any gene critical to chloroplast function (e.g. genes required for the photosynthetic apparatus) will result in reduced chloroplast production and decreased chlorophyll levels (Ishizaki et al., 2005;Ma et al., 2007;Waters and Langdale, 2009). To get direct insight into the function of the GNC/CGA1 family, we expressed GNC and CGA1 as GFP fusion proteins in stable transgenic plants using the CaMV 35S promoter, which results in high level, ubiquitous expression in Arabidopsis. The lines overexpressing either of these genes exhibited small dark-green cotyledons, as well as green hypocotyls and roots, the latter being tissues in which chlorophyll is not produced at high levels in wild-type plants under these growth conditions ( Fig. 2; Supplemental Fig. S5). The enhanced greening found in the CaMV 35Sdriven GNC and CGA1 lines indicates, even though some expression is normally observed in these tissues based on GUS-fusion analysis (Supplemental Fig. S1), that the native expression level of GNC/CGA1 family is limiting or that there are differences in regulation between the endogenous and ectopically produced transcription factors. Overexpression lines in which the genes were expressed without the GFP tag exhibited similar greening phenotypes, indicating that the fused GFP does not alter the function of these transcription factors (Supplemental Fig. S5). For the remainder of this study, we focused on two independent GNC overexpression lines (OX-1 and OX-2) because the loss-of-function analysis supports a more substantial role for GNC compared to CGA1 in greening.
However, CGA1 overexpression lines exhibited similar phenotypes to those found with GNC (Supplemental Figs. S5 and S6).
To further characterize the greening in cotyledons, hypocotyls, and roots, chlorophyll autofluorescence was examined by confocal microscopy. Interestingly, we observed chlorophyll fluorescence in the epidermal pavement cells of cotyledons, a celltype in which chloroplasts are typically not produced ( Fig. 2; Supplemental Videos S1 and S2). Furthermore, the normal jigsaw-puzzle shape of cotyledon pavement cells was changed to a more rounded mesophyll-like shape in the overexpression lines, indicating that overexpression of the GNC/CGA1 family may lead to an alteration of cell fate. These effects from the ectopic expression of GNC on chloroplast production and the shape of pavement cells have not previously been reported. Stronger chlorophyll fluorescence was detected in the hypocotyls of overexpression lines compared to those of wild type (Fig. 2).
In contrast, the gnc cga1 double mutant exhibited weaker chlorophyll fluorescence than the wild type (Fig. 2). We also observed that the hypocotyls of overexpression lines are longer compared to those of wild type and the gnc cga1 double mutant (Fig. 2). Significantly, the overexpression lines exhibited chlorophyll fluorescence in the roots whereas no fluorescence was detected in wild-type and the gnc cga1 double mutant roots (Fig. 2), consistent with chlorophyll biosynthesis normally being repressed in the roots under these growth conditions. In all tissues and cells examined, the chlorophyll fluorescence was punctate in appearance, consistent with its localization to chloroplasts.

Ectopic Overexpression of GNC or CGA1 Increases Chloroplast Biogenesis in the Hypocotyl
To determine whether the overexpression of GNC induces the biogenesis of functional chloroplasts, we examined the number and distribution of plastids in cross-sections of hypocotyls. We found two key differences between the overexpression lines and the wild type. First, chloroplast biogenesis in the cortical cell layers of the overexpression lines is significantly enhanced (Fig. 3, A and B). Quantification revealed that the chloroplast number per cell was increased more than two-fold in the cortical cells of overexpression lines compared to wild type or the gnc cga1 double mutant. The largest number of chloroplasts was found in the line OX-2 (Fig. 3B), which has the highest level of GNC protein (Fig. 3E), even though the transcript levels of GNC in the lines OX-1 and OX-2 were similar (Fig. 3D). There was no significant difference between wild type and the gnc cga1 double mutant in the number of chloroplasts in hypocotyl cortical cells (Fig. 3B).
These results are consistent with the analysis of chlorophyll content in the hypocotyls (Fig.   3C), in which the overexpression lines exhibited a ten-fold increase in chlorophyll levels compared to wild type, but there was no significant difference in chlorophyll levels between wild type and the gnc cga1 double mutant.
The second difference between the overexpression lines and wild type was in the production of chloroplasts in the hypocotyl epidermal cells. In wild type and the gnc cga1 double mutant, the average number in a cross-section is about 0.6 chloroplasts per epidermal cell (Fig. 3B), consistent with epidermal cells not normally producing fully developed chloroplasts (Dupree et al., 1991). In contrast, overexpression of GNC results in enhanced chloroplast biogenesis, with about four chloroplasts per epidermal cell in a cross-section, representing an increase of greater than six-fold. The ectopic production of chloroplasts in the hypocotyl epidermis is consistent with what we observed in the cotyledon pavement cells of the overexpression lines (Fig. 2), and indicates that ectopic expression of GNC expands the zones of chloroplast production. Cross-sections of hypocotyls from CGA1 overexpression lines revealed similar effects on the distribution and number of chloroplasts in cortical and epidermal cells as those seen in the GNC overexpression lines (Supplemental Fig. S6).

GNC and CGA1 Positively Regulate Chloroplast Growth in the Hypocotyl
We examined the ultrastructure of chloroplasts in the hypocotyls by transmission electron microscopy (TEM). Interestingly, in spite of there being no significant difference in hypocotyl chloroplast number between wild type and the gnc cga1 double mutant (Fig. 3B), the size of chloroplasts differs (Fig. 4, A and B). The cross area of chloroplasts in the cortical cells showed a 27 percent reduction in the gnc cga1 double mutant whereas chloroplasts in the overexpression lines were over two-fold larger than those in the wild type ( Fig. 4B). It should be noted, however, that the chloroplasts in the hypocotyls of overexpression lines are not larger than those in the wild-type leaf (5-10 µm in diameter) (López-Juez and Pyke, 2005), suggesting that GNC overexpression enhanced chloroplast growth in the hypocotyls where chloroplast development is normally retarded. These results demonstrate that GNC and CGA1 positively regulate chloroplast growth. In spite of the smaller chloroplasts in the gnc cga1 double mutant, no obvious defects of the thylakoid membranes or grana were observed (Fig. 4A). In addition, chloroplasts from the gnc cga1 double mutant and overexpression lines produced starch granules resembling those of wild-type chloroplasts (Fig. 4A). Photosynthetic efficiency analysis showed that the maximum quantum yield of photosystem II (Fv/Fm) and the flux of electrons through photosystem II (Φ PSII ) in the hypocotyls of overexpression lines is similar to those in the leaves of wild type, the gnc cga1 double mutant, and the overexpression lines, indicating that the chloroplasts from all these tissues exhibit normal photosynthetic activity (

GNC Overexpression Promotes Chloroplast Biogenesis in the Root
Because the overexpression lines exhibit chlorophyll fluorescence in the roots, we examined the distribution of plastids in root cross-sections. The most pronounced difference between the GNC overexpression lines as compared to wild type and the gnc cga1 mutant was in the proliferation of chloroplasts in the pericycle and cortex. The ability of the root pericycle to produce chloroplasts has been previously noted (López-Juez, 2007); we observed a few chloroplasts in the wild type and gnc cga1 mutant pericycle, but these were reduced in number, smaller, and with fewer thylakoid membranes than found in the overexpression lines. Interestingly, we did not observe any proliferation of chloroplasts within the endodermis following overexpression of GNC, although chloroplasts were occasionally noted in the wild-type endodermis (one or two chloroplasts per cross section).

GNC Overexpression Enhances Chloroplast Development in the Dark
Light is normally a prerequisite for chlorophyll biosynthesis and the completion of chloroplast development (Armstrong et al., 1995;Waters and Langdale, 2009). To determine whether overexpression of GNC enhances chloroplast development in the dark, we examined plastid morphology in the hypocotyls of dark-grown seedlings by TEM.
Hypocotyls were used as we observed pronounced effects of GNC on chloroplast production in this tissue. In 7-day-old dark-grown seedlings, proplastids were not yet differentiated into plastids either in the wild-type or in the gnc cga1 double mutant hypocotyls (Fig. 6A). In contrast, most plastids in the hypocotyls of the overexpression lines had transformed into etioplasts with their distinctive prolamellar bodies and prothylakoids (Fig. 6A).
To examine whether the etioplasts in the overexpression lines are readily converted to mature chloroplasts, we performed a light-induced de-etiolation experiment. For this purpose, seven-day-old dark-grown seedlings were transferred to white light for different periods of time. Before illumination, the seedlings displayed etiolated phenotypes, with closed yellow cotyledons and elongated hypocotyls (Fig. 6B). After 24-hour light exposure, wild-type seedlings showed yellow cotyledons with newly emerging green true leaves while the gnc cga1 double mutant only exhibited yellow cotyledons. Interestingly, unlike wild type and the gnc cga1 double mutant, the overexpression lines exhibited expanded green cotyledons with newly emerging green true leaves within 24 hours after illumination.
The strongest response was found in OX-2 with the highest GNC protein level.
To determine the immediate effect of the loss-and gain-of-function mutations on de-etiolation, dark-grown seedlings were moved to light for six hours and de-etiolation was assessed by measuring chlorophyll levels in the seedlings (Fig. 6C). The gnc cga1 mutant exhibited reduced chlorophyll levels compared to wild type. Significantly, the overexpression lines exhibited higher accumulation of chlorophyll than wild type. These results suggest that overexpression of GNC enhances greening by promoting the development of etioplasts from proplastids in the dark and that these etioplasts are promptly converted to chloroplasts upon illumination.

Increases in Chloroplast Biogenesis Can Be Regulated Post-Embryonically
Many of the greening phenotypes we and others have associated with ectopic expression of the GNC/CGA1 family occur in the cotyledon, hypocotyl, and root (Richter et al., 2010;Hudson et al., 2011;Köllmer et al., 2011), all of which are embryonically derived. This is of significance because chloroplasts are present in these tissues during the early stages of embryo development, including being found within the epidermal cell layer (Tejos et al., 2010). During later stages of embryo development, chloroplasts undergo de-differentiation to form basal-state plastids as seeds develop. These basal-state plastids can then redifferentiate to chloroplasts or other plastids upon germination, suggesting that postembryonic factors in concert with embryonic factors regulate chloroplast differentiation during plant development (Whatley, 1978;Zhao and Sack, 1999). Thus, a possible explanation for the increased levels and expanded zones of chloroplast production is that overexpression of GNC/CGA1 family prevents the chloroplasts in the embryos from the de-differentiation or increases levels of some embryonically-derived factor such that plastids are more likely to take on a chloroplast identity following germination.
To determine whether the striking phenotypes in the embryonically-derived tissues require the expression of GNC during embryogenesis, we generated transgenic lines in a gnc cga1 mutant background in which the expression of GNC can be induced by the application of a steroid hormone dexamethasone (DEX) (Aoyama and Chua, 1997). The induction of GNC by DEX treatment was confirmed by a 4-h induction period with 10 µM DEX, following which we observed induction of GNC in the DEX-inducible line  to twice the wild-type level; no detectable expression of GNC occurred in the absence of DEX treatment (Fig. 7A). To examine chloroplast production, DEX-inducible GNC seedlings were grown on the plates containing 1 µM DEX or a DMSO vehicle control under constant white light for 7 days. DEX-L7 seedlings exhibit long green hypocotyls only when DEX is supplemented on the plates (Fig. 7B), the same phenotype we observe when GNC is ectopically expressed (Fig. 2). Significantly, we also observed chlorophyll

Chloroplast Number in Cotyledons and Leaves
The cotyledons, hypocotyls, and roots of seedlings over-expressing GNC or CGA1 were darker green than those of wild type, but the cotyledons of the over-expression lines were smaller than those of wild type (  S9). Chloroplasts were observed in the epidermal pavement cells of leaves when GNC was ectopically expressed from either the CaMV 35S promoter or following induction from a DEX-inducible promoter, although at substantially reduced levels compared to what we observed in the epidermal pavement cells of cotyledons (Fig. 2).

Effect of Cytokinin on Chloroplast Division in Mutants of the GNC/CGA1 Family
Cytokinin stimulates chloroplast division in cotyledons, resulting in a smaller size but increased numbers of chloroplasts (Okazaki et al., 2009). We therefore examined the effects of cytokinin on chloroplast size and number in wild type, the gnc cga1 mutant, and the GNC overexpression lines (Fig. 8, A and B). Growth of wild type with exogenous BA results in a reduction in chloroplast size, such that at 10 µM BA the chloroplast crosssectional area in wild type is approximately 50% of that found in untreated seedlings (Fig.   8A). Chloroplast size of the gnc cga1 mutant is similar to that of wild type in the untreated sample, but exhibits reduced sensitivity to cytokinin, resulting in significantly larger chloroplasts than wild type at all cytokinin concentrations examined ( Expression of CGA1 and potentially also of GNC is positively regulated by cytokinin through action of the type-B ARRs, in particular ARR1 and ARR12 (Fig. 1). We therefore examined whether the cytokinin-mediated regulation of chloroplast division also requires ARR1 and ARR12 by examining the cytokinin responsiveness of the arr1-3 arr12-1 double mutant for this phenotype (Fig. 8, C  decrease in size and increase in number when grown of 1 µM BA, the arr1-3 arr12-1 mutant exhibits no difference in chloroplast size or number following cytokinin treatment, indicating that the type-B ARRs are required for cytokinin effects on chloroplast division. These results support a general role for the GNC/CGA1 family in chloroplast division and, more specifically, indicate that cytokinin mediates its effects on chloroplast division through GNC and CGA1 in a type-B ARR-dependent manner. The finding that the gnc cga1 mutant still exhibits some response to cytokinin indicates that other factors, such as the CRF family of transcription factors (Okazaki et al., 2009), functionally overlap with the GNC/CGA1 family in this response.

DISCUSSION
In this study, we characterized the role of GNC and CGA1 in plant development, focusing on several aspects of chloroplast biogenesis. Our study contributes novel evidence supporting roles for GNC and CGA1 in mediating the development of chloroplasts from proplastids, in enhancing chloroplast growth and division, and in serving as targets for cytokinin regulation of these processes. Below we discuss our results within the context of (1) chloroplast biogenesis, (2) cytokinin signaling, and (3) the overall transcriptional control of chloroplast development. A model situating the GNC/CGA1 family within this regulatory context is given in Figure 9.

Key Role of the GNC/CGA1 Family in Chloroplast Biogenesis
Our results demonstrate that the GNC/CGA1 family regulates multiple aspects of  family (Mara and Irish, 2008). However, we did not observe greening in the petals of overexpression lines, indicating that other factors required for greening are absent or that the activity of the GNC/CGA1 family is regulated post-transcriptionally.
The GNC/CGA1 family not only regulates the development of chloroplasts from proplastids but also, once the chloroplasts are formed, regulates their growth and division.
A role in chloroplast growth is supported by the smaller chloroplasts found in hypocotyl cortex cells of the gnc cga1 mutant compared to wild type, even though the absolute number of chloroplasts did not differ in the cells. Also consistent with an effect on growth is the finding that overexpression of GNC and CGA1 results in the opposite phenotype, chloroplasts of the hypocotyl cortex cells being larger than those found in wild type. A role in chloroplast division is supported by our analysis of cytokinin-regulated chloroplast division in cotyledons. Evidence that division is altered in other tissues comes from our finding that overexpression of GNC and CGA1 results in increased numbers of chloroplasts in epidermal and cortical cells of the hypocotyl, as well as the finding that GNC/CGA1 mutants alter the numbers of chloroplasts in leaves (Hudson et al., 2011). Chloroplasts multiply by binary fission (Possingham and Lawrence, 1983;Kuroiwa et al., 1998), and thus an increase in chloroplast number indicates an acceleration of chloroplast division.

Role of the GNC/CGA1 Family in Cytokinin Signaling
There is a long history of research implicating cytokinins in the control of chloroplast  Yaronskaya et al., 2006), and enhance chloroplast division (Okazaki et al., 2009). Genetic analysis of the cytokinin signal transduction pathway is also consistent with cytokinin regulating chloroplast development, because mutation of the cytokinin receptors (ahk2 ahk3 ahk4 triple mutant) or the type-B ARRs (arr1 arr10 arr12 triple mutant) result in reduced chlorophyll levels (Riefler et al., 2006;Argyros et al., 2008). In spite of the significance of cytokinin for chloroplast biogenesis, the molecular mechanism of its action on chloroplasts is largely unknown. Our results suggest that the GNC/CGA1 family serves as one of the transcriptional outputs by which cytokinin modulates chloroplast development (Fig. 9). The GNC/CGA1 family also functions after chloroplast development in the regulation of chloroplast growth and division, and at this point appears to overlap with the function of the CRF family (Fig. 9). Unlike GNC/CGA1 overexpression lines, neither ectopic production of chloroplasts nor increased chlorophyll content has been reported from CRF overexpression lines (Okazaki et al., 2009). However, like the GNC/CGA1 overexpression lines, overexpression of CRF2 mimics the effect of cytokinin on chloroplast division, resulting in increased numbers of smaller chloroplasts (Okazaki et al., 2009).
Based on their roles in the regulation of chloroplast biogenesis, it is perhaps not too surprising that expression of these transcription factors is regulated by key signals implicated in the control of chloroplast development, in particular light and cytokinin (Fig.   9). The GNC/CGA1 and GLK families are both transcriptionally up-regulated by light (Fitter et al., 2002;Naito et al., 2007), supporting an increased role under conditions where chloroplasts are actively produced. In addition, both the GNC/CGA1 and CRF families are transcriptionally up-regulated by cytokinin (Rashotte et al., 2006;Naito et al., 2007). We find that CGA1 is strongly and transiently up-regulated in response to treatment with exogenous cytokinin. GNC is less responsive to exogenous cytokinin but, like CGA1, exhibits reduced expression in a type-B arr mutant background, consistent with the expression of both being affected by changes in endogenous cytokinin signal output.
Although the expression of the GLK family does not appear to be regulated by cytokinin, the GLK transcription factors have a DNA-binding domain similar to that of type-B ARRs (Fitter et al., 2002), implying that their overlapping role in chloroplast biogenesis may be mediated in part by both binding to similar promoter sequences. Taken together, these data support a transcriptional network in which the GNC/CGA1 family acts in concert with at least two other families of transcription factors to control chloroplast development, growth, and division, and is responsive to inputs by both light and cytokinin to modulate these processes.

Plant Material and Growth Conditions
T-DNA insertion lines for the genes GNC (SALK_001778) and CGA1 (SALK_003995) were obtained from the Arabidopsis Biological Resource Center (Bi et al., 2005;Naito et al., 2007;Mara and Irish, 2008). The double gnc cga1 mutant was generated by crossing.
T-DNA insertion mutants were genotyped by PCR using T-DNA left border and the genespecific primers as listed in Table S1.
Unless stated otherwise, seeds were surface-sterilized and stratified for 3 days in the dark at 4˚C, before being moved into the light. Seedlings for molecular and physiological assays were grown on medium containing 0.8% (w/v) phytoagar (Research Arabidopsis by the floral dip method (Clough and Bent, 1998).

Histochemical Analysis of GUS Activity
Histochemical analysis of GUS activity in stably transformed lines of Arabidopsis was performed as described (Argyros et al., 2008). Embryo GUS staining was performed as described with the following modification (Yadegari et al., 1994) with the following modification. Siliques were opened longitudinally and then incubated with GUS staining solution at 37˚C overnight. Siliques were then fixed in an ethanol:acetic acid solution (1:1) for 3 hours and cleared in a chloral hydrate:glycerol:water solution (8:1:2 ; w:v:v) overnight. Dissected ovules were mounted in 50% glycerol with a few drops of chloral hydrate solution and embryos removed from the ovules by applying pressure with needles.
The GUS-stained tissues were visualized and photographed using an EPSON scanner or Zeiss Axioplan 2 microscope under bright field conditions.
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Gene Expression Analysis
RNA isolation and qRT-PCR analyses were performed as described (Argyros et al., 2008).
Briefly, total RNA was isolated by using the RNeasy plant kit according to the manufacturer, with the incorporation of a DNase treatment (QIAGEN, Valencia, CA). First strand cDNA synthesis for RT-PCR and qRT-PCR were performed using SuperScript III with oligo-dT primers (Invitrogen, Carlsbad, CA). For RT-PCR, gene-specific primers (Table S1) were used to examine the full-length transcripts of GNC or CGA1 with HS-Taq polymerase (TaKaRa Bio Inc, Otsu, Shiga, Japan). qRT-PCR was performed using SYBR Premix Ex Taq II (TaKaRa Bio Inc, Otsu, Shiga, Japan) and the primer sets listed in Table   S1. Average Ct values were generated and analyzed by SDS software v.1.4, which uses the comparative CT method (Livak and Schmittgen, 2001). β-tubulin 3 (β-TUB 3) (AT5G62700) was used as control for both RT-PCR and qRT-PCR.

Immunoblot Analysis
Ten-day-old seedlings were ground in liquid nitrogen and treated with homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 0.1% Nonidet P-40, and plant protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The mixture was centrifuged at 12,000 g for 15 minutes, the supernatant isolated, and protein concentration determined using the BCA protein assay (Thermo scientific, Waltham, MA).
Proteins were separated on 10% SDS-PAGE gels, and transferred to PVDF membranes as described (Gamble et al., 2002). GFP-fusion proteins were detected with an HRPconjugated monoclonal anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
The loading control α-Tubulin was detected with a mouse monoclonal anti-tubulin antibody (Sigma-Aldrich, St. Louis, MO) and a secondary goat anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Chlorophyll Measurements
Total chlorophyll levels were determined by extraction of plant material with 95% (v/v) ethanol at 4°C in the dark and shaking for 16 hours, with spectrophotometric determination of chlorophyll content made as described (Lichtenthaler, 1987)  For de-etiolation, seeds were stratified and illuminated under 100 µmol m -2 s -1 white light for 1 hour to coordinate germination. Seedlings were grown on 1X MS media for 5 days in the dark and illuminated with 100 µmol m -2 s -1 white light for 6 hours to induce deetiolation. Chlorophyll content was measured as described (Lichtenthaler, 1987

Chloroplast Visualization
For visualization of chloroplasts by autofluorescence in living tissues, hypocotyls from 7day-old light-grown seedlings and roots from 10-day-old light-grown seedlings were excised, stained with propidium iodide (10 µg/ml), mounted in water, and visualized using a Leica TCS SP UV confocal microscope at an excitation wavelength of 488 nm.
Chlorophyll autofluorescence was detected between 631 nm and 729 nm. Propidium iodide emission was detected between 562 nm and 612 nm. Image series taken in the z plane were processed by Imaris (Bitplane Scientific Software, Zurich, Switzerland) to obtain the maximum intensity projections. For visualization in cotyledons, 14-day-old light grown seedlings were used, being mounted adaxial side uppermost in water, and visualized using a Nikon A1 confocal microscope at the excitation and emission wavelengths given above.
For visualization of chlorophyll autofluorescence in the palisade mesophyll cells of leaves, a Nikon A1 confocal microscope was used with quantitative analysis of chlorophyll autofluorescence performed using NIS-Elements AR Analysis 4.00.03 software.
For visualization of chloroplasts autofluorescence from DEX-inducible lines, cotyledons from 7-day-old seedlings grown in the presence or absence of 1 µM DEX were used. Samples were prepared as described above and mounted abaxial side uppermost in water, and visualized using a Nikon A1 confocal microscope at the excitation and emission wavelengths given above.

For visualization of chloroplasts in the palisade mesophyll cells of leaves, a Zeiss
Axioplan 2 microscope was used. The area of the chloroplasts as well as of the palisade cells was measured using ImageJ software (version 1.32, National Institute of Health, Bethesda, MD).
For visualization of chloroplasts in fixed tissues, 1 mm-long hypocotyl segments at the root-hypocotyl junction were dissected from 7-day-old light-grown seedlings, 2 mmlong hypocotyl segments were dissected under dim green light from 4-day-old dark-grown seedlings, and 2 mm-long root segments were dissected at 5 mm away from the roothypocotyl junction of 7-day-old light-grown seedlings. Preparation of the samples was performed as described (Härtel et al., 1998) (ΦPSII) were calculated as described (Maxwell and Johnson, 2000).

Cytokinin Effects on Chloroplast Division
For chloroplast size measurement, chloroplasts were isolated from the cotyledon tips of 11day-old seedlings grown on 1X MS media supplied with 1% (w/v) sucrose and the

Supplemental Data
The following materials are available in the online version of this article: Supplemental Figure S1. Tissue specific expression of GNC and CGA1.
Supplemental Figure S2. GNC and CGA1 transcription factors localize to the nucleus.
Supplemental Figure S3. Characterization of the gnc and cga1 T-DNA insertion alleles.
Supplemental Figure S4. Altered levels of chlorophyll in the gnc cga1 mutant.
Supplemental Figure S5. Overexpression of GNC and CGA1 induces chloroplast biogenesis in the hypocotyls.
Supplemental Figure S6. Overexpression of GNC and CGA1 results in increased chloroplast number, and expanded zones of chloroplast production.
Supplemental Figure S8. Chlorophyll a content in cotyledons of the gnc cga1 mutant and GNC overexpression lines is altered compared to wild type.
Supplemental Figure S9. Effect of gnc cga1 mutation and GNC overexpression on rosette weight and chlorophyll accumulation in leaves.
Supplemental Figure S10. Effect of gnc cga1 mutation and GNC overexpression on chloroplast autofluorescence, size of mesophyll cells, chloroplast size, and number of chloroplasts per cell.
Supplemental Table S1. DNA primers used in this study    used as the internal control. E, Total protein extracted from 10-day-old seedlings was analyzed by anti-GFP antibodies. α-tubulin as a loading control was detected by antitubulin antibodies.