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Keywords:

  • gibberellin;
  • DELLA proteins;
  • chloroplast biogenesis;
  • chloroplast division;
  • cell expansion;
  • grana stacking

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Chloroplast biogenesis needs to be well coordinated with cell division and cell expansion during plant growth and development to achieve optimal photosynthesis rates. Previous studies showed that gibberellins (GAs) regulate many important plant developmental processes, including cell division and cell expansion. However, the relationship between chloroplast biogenesis with cell division and cell expansion, and how GA coordinately regulates these processes, remains poorly understood. In this study, we showed that chloroplast division was significantly reduced in the GA-deficient mutants of Arabidopsis (ga1-3) and Oryza sativa (d18-AD), accompanied by the reduced expression of several chloroplast division-related genes. However, the chloroplasts of both mutants exhibited increased grana stacking compared with their respective wild-type plants, suggesting that there might be a compensation mechanism linking chloroplast division and grana stacking. A time-course analysis showed that cell expansion-related genes tended to be upregulated earlier and more significantly than the genes related to chloroplast division and cell division in GA-treated ga1-3 leaves, suggesting the possibility that GA may promote chloroplast division indirectly through impacting leaf mesophyll cell expansion. Furthermore, our cellular and molecular analysis of the GA-response signaling mutants suggest that RGA and GAI are the major repressors regulating GA-induced chloroplast division, but other DELLA proteins (RGL1, RGL2 and RGL3) also play a role in repressing chloroplast division in Arabidopsis. Taken together, our data show that GA plays a critical role in controlling and coordinating cell division, cell expansion and chloroplast biogenesis through influencing the DELLA protein family in both dicot and monocot plant species.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Chloroplasts are essential for photosynthesis, for the production of amino acids, lipids and phytohormones, and for the storage of starch and oil compounds (Sakamoto et al., 2008). Chloroplast biogenesis is initiated from proplastids in small meristematic cells, and involves a series of important events that bring about the development of the organelle from a simple proplastid to the photosynthetically active chloroplast, with coordinated changes in organelle size, thylakoid membrane abundance and composition, pigment accumulation, organelle division and chloroplast genome copy number (Biswal et al., 2003; Stern et al., 2004). Chloroplast division involves the formation of a ring structure at the site of division (Okazaki et al., 2009). Recent molecular genetic studies in Arabidopsis have identified several proteins that are the components of the chloroplast division ring, including: FtsZ1 and FtsZ2, self-assembling tubulin-like GTPases (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Mori et al., 2001; Vitha et al., 2001; Kuroiwa et al., 2002); ARC5 and ARC6, a member of the eukaryotic membrane-remodeling GTPases and a regulator of Z-ring assembly and dynamics, respectively (Gao et al., 2003; Yang et al., 2008); and PDV1 and PDV2, plant-specific components of the plastid division ring (Okazaki et al., 2009). Transcriptional or post-translational reductions in the levels of chloroplast division ring components result in the significant suppression of the chloroplast division rate (Sakamoto et al., 2008; Yang et al., 2008).

In higher plants, chloroplast division needs to be coordinated with cell division and cell expansion during leaf growth and development to maintain a relatively stable number of chloroplasts per cell for the efficient capture of light energy (Pyke, 1999). It has been suggested that the size, but not the ploidy, of a leaf mesophyll cell is the primary determinant of the chloroplast number within the cell (Pyke and Leech, 1992). During mesophyll cell expansion, the chloroplast number per cell is tightly correlated with the cell size (Pyke and Leech, 1992). Moreover, increased chloroplast divisions were found in response to artificially increased cell size (Pyke, 1997). Recently, several studies suggested that plastid division and cell division are partially regulated by common pathways. For example, a deficiency in AtCRL, which encodes a protein localized in the envelope membrane of the plastids, affected the orientation of the cell division plane and displayed few but enlarged chloroplasts within the mesophyll cells (Asano et al., 2004). Downregulation of the pre-replication factor AtCDT1 is shown to slow down both the cell cycle and plastid division in Arabidopsis (Raynaud et al., 2005). However, it is still poorly understood how cell expansion, cell division and chloroplast division are coordinately regulated at the molecular and cellular levels.

Gibberellin (GA) is a key plant growth hormone that plays an essential role in regulating many aspects of plant growth and development, such as seed germination, leaf expansion, stem elongation and floral induction (Kende and Zeevaart, 1997; Biemelt et al., 2004). GA regulates growth by promoting the degradation of a family of repressive DELLA proteins, which are encoded by a family of five genes (RGA, GAI, RGL1, RGL2 and RGL3; Dill and Sun, 2001; King et al., 2001; Peng et al., 1997; Silverstone et al., 1998). Accumulating evidence is suggesting that GA also plays an important role in regulating chloroplast biogenesis and photosynthesis. Firstly, GAs prevent Arabidopsis seedling de-etiolation (greening) in darkness by modulating light signaling pathways (Alabadi et al., 2008). Secondly, GA-regulated DELLA proteins play a key role in the formation of functional young chloroplasts during de-etiolation, by regulating the levels of the photoprotective enzyme protochlorophyllide oxidoreductase (POR), protochlorophyllide and carotenoids in the dark, which is important in protecting the etiolated seedlings against photooxidative damage during initial light exposure (Cheminant et al., 2011). Thirdly, GA plays a positive role in the control of photosynthesis in mature leaf tissue of Solanum lycopersicum (tomato), Nicotiana tabacum (tobacco) and Carrizo citrange (orange) plants (Arteca and Dong, 1981; Biemelt et al., 2004; Huerta et al., 2008). These data suggest that GAs are involved in the control of young chloroplast development, and play a positive role in enhancing the photosynthetic activity of mature chloroplasts. However, it remains unknown how GA regulates chloroplast division and thylakoid organization, two vital events of chloroplast biogenesis for light capture during photosynthesis.

In this study, we employed leaf anatomy and quantitative reverse transcriptase PCR to examine the phenotypes of chloroplast biogenesis and mesophyll cell development in the GA-deficient mutants ga1-3 of Arabidopsis and d18-AD of rice. Our data indicated that GA plays a positive, but probably indirect, role in the regulation of chloroplast division, and plays an important role in coordinating chloroplast division, cell expansion and/or cell division during leaf development. Moreover, an analysis of the GA-response mutants gai-1, rga-24 gai-t6 and della (a pentuple mutant of all five DELLA genes) suggests that chloroplast division and grana stacking are regulated via a DELLA-dependent GA-signaling pathway in Arabidopsis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Gibberellin affects leaf cell division, expansion and organization

To investigate the roles of GA in the regulation of leaf development, we examined the leaf morphology and anatomy in ga1-3 and wild-type plants. A significant decrease in leaf area (by 89.6%) but increased pigmentation was found in ga1-3 compared with the wild type (Figure 1a). An investigation into the anatomical features of the leaf showed that the ga1-3 mutant exhibited more tightly packed mesophyll cells and thicker mesophyll cell layers (Figure 1b), corresponding with an increase of mesophyll cell number per unit leaf area (by 42.6%; Figure 1c) and a decrease of mesophyll cell area (by 42.8%; Figure 1d), compared with the wild type. Moreover, a decrease of cell number per leaf (by 75.1%) was found in ga1-3 compared with the wild type (Figure 1e). Application of exogenous GA largely restored the wild-type leaf anatomy phenotypes in the ga1-3 mutant (Figure 1b). These results indicate that GA plays a critical role in regulating cell division, cell expansion and organization during leaf growth and development.

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Figure 1.  Gibberellin (GA) affects leaf cell division, expansion and organization. Morphological and anatomical study of 3-week-old wild-type and ga1-3 mutant plants, with or without exogenous GA treatment. (a) Approximately 3-week-old wild-type and ga1-3 mutant plants used for this study (scale bar: 1 cm). L4 and L5 indicate the fourth and fifth leaves used for the experiments, respectively. Numbers indicate the average leaf area of the fourth leaf (cm2) (= 3 leaves, ±SD). *Statistically significant differences assessed by Student’s t-tests (< 0.01). SD, standard deviations. (b) Cross sections of the center of the fourth leaf of the wild type and ga1-3 plants (scale bar: 50 μm). (c) Statistical comparison of cell number per unit leaf area in the center of the fourth leaf (= 3 leaves, ±SD) of wild-type and ga1-3 plants. *Statistically significant differences, assessed by Student’s t-tests (< 0.05). (d) Statistical comparison of the mesophyll cell area (= 39 cells, ±SD) of wild-type and ga1-3 plants. *Statistically significant differences, assessed by Student’s t-tests (< 0.01). (e) Comparison of cell number per leaf (fourth leaf, = 3 leaves, ±SD) of wild-type and ga1-3 plants. (f) Statistical comparison of chloroplast no. per unit leaf area in the center of the fourth leaf (= 3 leaves, ±SD) of wild-type and ga1-3 plants. *Statistically significant differences, assessed by Student’s t-tests (< 0.01). (g) Comparison of the chlorophyll content per unit leaf area (1 cm2) (= 3 leaves, ±SD) of wild-type and ga1-3 plants. (h) The rate of photosynthetic oxygen evolution from unit leaf area (2.31 cm2) of the fourth leaves of wild-type and ga1-3 plants. A representative experiment is shown. Two biological replicates gave the same result.

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To identify the underlying causes for the increased pigmentation in ga1-3, we examined the chloroplast number per unit leaf area, which was counted from the cross sections of the fourth leaves (representing expanding leaves). An increase in the chloroplast number per unit leaf area (by 48.9%) was found in ga1-3 (Figure 1f), corresponding with an increase in chlorophyll content per unit leaf area (by 146.1%; Figure 1g), suggesting that the increased pigmentation in the ga1-3 mutant can be attributed to an increase in chloroplast number per unit leaf area. Moreover, an increased rate of photosynthesis, which was assessed by oxygen evolution rate (Figure 1h and Appendix S1), was found in ga1-3 compared with the wild type, suggesting that the dwarfed GA-deficient mutants may maintain a proper level of photosynthetic activity by possessing higher chlorophyll content per unit leaf area.

GA deficiency altered the relationships of chloroplast number per cell and mesophyll cell area

To determine whether the decrease in mesophyll cell area (Figure 1c) has an impact on the chloroplast number per cell in the ga1-3 mutant, we investigated the number of chloroplasts per fully expanded palisade mesophyll cell in ga1-3. Surprisingly, we found no significant difference when compared with the wild type (Figure 2a), suggesting that the chloroplast density per cell is increased in ga1-3. To confirm this, we compared the relationships between chloroplast number per cell and mesophyll cell area in ga1-3 and the wild type by using a best-fit linear plot of chloroplast number per cell versus cell area. The slope of the best-fitting line for ga1-3 (0.036) is steeper than that for the wild type (0.021; Figure 2b), corresponding with an increased number of chloroplasts per unit cell area in ga1-3 (by 67.9%), compared with the wild type (Figure 2c). These results indicate that the relationship between chloroplast number and cell size is radically altered by GA deficiency.

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Figure 2.  Gibberellin (GA) deficiency alters the relationships between chloroplast number per cell and mesophyll cell size. (a) Chloroplasts in the isolated mesophyll cells from the center of the fourth leaf of wild-type and ga1-3 plants (scale bar: 10 μm). Numbers indicate the average chloroplast number per mesophyll cell (= 39 cells, ±SD). The value of ga1-3 is not significantly different from the wild type when assessed by a Student’s t-test (> 0.05). SD: standard deviations. (b) The relationship between chloroplast number per mesophyll cell and mesophyll cell plan area for wild-type and ga1-3 plants. Each data point represents the measurement from one mature mesophyll cell, for which the area was measured and the chloroplast number was counted (= 39 cells). The best-fitting line for each relationship is shown. The slopes for each best-fitting line are 0.021 (wild type) and 0.036 (ga1-3). (c) Statistical comparison of the number of chloroplasts per unit cell plan area for the fourth leaf of wild-type and ga1-3 plants (= 39 cells, ±SD), with or without exogenous GA treatment. *Statistically significant differences, assessed by Student’s t-tests (P < 0.01).

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GA promotes chloroplast division in Arabidopsis

To test whether chloroplast division is affected in ga1-3, we estimated the chloroplast number per leaf in ga1-3 and in the wild type. A decreased chloroplast number per leaf (by 72.8%; Figure 3a) is consistent with a decreased chloroplast division frequency (measured as the percentage of dumbbell-shaped chloroplasts per random selected chloroplasts in leaf tips; n ≥ 350 chloroplasts; by 49.8%) in the ga1-3 mutant relative to the wild type (Figure 3b). The application of exogenous GA approximately restored wild-type chloroplast division traits in the ga1-3 mutant (Figure 3a,b). These observations indicate that GA promotes chloroplast division in leaf mesophyll cells.

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Figure 3.  Gibberellins (Gas) are involved in the regulation of chloroplast division in Arabidopsis. (a) Comparison of the number of chloroplasts per leaf (fourth leaf, = 3 leaves) of wild-type and ga1-3 plants, with or without exogenous GA treatment. (b) Statistical comparison of chloroplast division frequency in the fourth leaf tip (≥ 350 chloroplasts) of wild-type and ga1-3 plants, with or without exogenous GA treatment. *Statistically significant differences, assessed by Student’s t-tests (< 0.01). (c) Transcript levels of selected chloroplast division genes in the fifth leaf of wild-type and ga1-3 plants, with or without exogenous GA treatment, were determined by quantitative RT-PCR. The error bars represent SDs (standard deviations).

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We next performed quantitative RT-PCR to measure the transcript levels of genes encoding structural components of the chloroplast division apparatus (Table S3 for the gene-specific primers). The transcript levels of AtPDV1, AtPDV2, AtARC5, AtARC6 and AtFtsZ2-1 were significantly reduced in ga1-3 relative to the wild type (Figure 3c). The application of exogenous GA restored the expression of chloroplast division genes in the ga1-3 mutant to approximately wild-type levels (Figure 3c), suggesting that GA also promotes the expression of chloroplast division-related genes.

GA coordinately promotes cell expansion, cell division and chloroplast division

As the decreased chloroplast number per leaf (by 72.8%; Figure 3a) was in parallel with the decreased cell number per leaf (by 75.1%) in ga1-3 compared with the wild type (Figure 1e), we next examined how GA coordinately regulates chloroplast division, cell division and cell expansion. To this end, we monitored the time-course changes for chloroplast division, cell division and cell expansion phenotypes during the treatment of exogenous GA on the ga1-3 mutant. Our results showed that GA treatment increased chloroplast number per cell (Figure 4a), chloroplast division frequency (Figure 4b), cell division rate (Figure 4d) and mesophyll cell area (Figure 4f) in ga1-3, suggesting that chloroplast division, cell division and cell expansion is coordinately promoted by GA.

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Figure 4.  Gibberellin (GA) promotes chloroplast division, cell expansion and cell division of ga1-3 mesophyll cells. Kinematic analysis of chloroplast division, cell division and cell expansion of the first true leaves of 2-week-old ga1-3 seedlings, treated with GA or water (control) at each time point. (a) Chloroplasts in the isolated mesophyll cells from the center of the leaves of ga1-3 plants (scale bar: 10 μm) treated with GA for the times, as indicated, and a control. Numbers indicate the average chloroplast numbers per mesophyll cell (= 20 cells, ±SD). SD, standard deviations. (b) Average (> 200 chloroplasts, ±SD) chloroplast division frequency in the first true leaf. (c, e and g) Relative mRNA levels of chloroplast division genes, cell division genes and expansin genes were determined by quantitative RT-PCR at each time point. The error bars represent SDs. (d) Average cell division rates in the first true leaves (= 3 leaves). (f) Average mesophyll cell area (= 20 cells, ±SD) in the first true leaf.

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Many studies showed that GA plays key roles in the control of cell expansion and cell division (Kende and Zeevaart, 1997). GA promotes cell expansion by regulating the behaviour of microtubules in the growing cells, corresponding with the upregulation of the gene family members of expansin, which are key regulators of wall extension during growth (Cho and Cosgrove, 2000; Wenzel et al., 2000; Ogawa et al., 2003; Sauret-Gueto et al., 2012). On the other hand, GA controls cell division by stimulating the expression of some cyclin and CDK genes to activate the G1/S and the G2/M transitions in rice (Sauter, 1997; Fabian et al., 2000). We next performed quantitative RT-PCR to measure the transcript levels of the genes related to chloroplast division, cell division and cell expansion. Our results showed that the majority of transcriptional upregulation for selected chloroplast division genes and cell division genes occurred between hours 24 and 48 (Figure 4c,e), whereas the transcriptional upregulation for expansin genes AtEXPA1 and AtEXPA8 occurred between hours 0 and 2 (Figure 4g), suggesting that the cell expansion-related genes tended to be upregulated much earlier and more significantly than genes related to chloroplast division and cell division.

GA regulates chloroplast grana stacking

To investigate whether GA affects the organization of the thylakoid network of chloroplasts, we examined the ultrastructure of the chloroplasts in the top layers of mesophyll cells of the fourth leaf in the ga1-3 mutant and the wild type, to avoid the shading effects on chloroplasts from upper cell layers. An increase in grana stacking (thylakoid membrane layers per granal stack) was found in ga1-3 (Figure 5a; Table S1). Wild-type granal stacking traits were restored by the application of exogenous GA to ga1-3, and the application of exogenous GA to wild type plants results in a decrease in the number of thylakoid membranes per granum (Figure 5a; Table S1). These results suggest that chloroplast grana stacking is also regulated by GA.

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Figure 5.  Changes in gibberellin (GA) levels affect grana stacking and photooxidative stress tolerance in Arabidopsis. (a) Ultrastructure of the chloroplasts in the top layers of mesophyll cells in the fourth leaf of wild-type and ga1-3 plants, with or without exogenous GA treatment. Scale bars: 1 μm (1, 2, 3 and 4) and 0.17 μm (5, 6, 7 and 8). (b) Maximal photochemical efficiency parameter (Fv/Fm) in the fourth leaf (= 3 leaves, ±SD) of wild-type and ga1-3 plants, during high-light stress (150 min, 1500 μmol m−2 sec−1), and after 300 min in recovery in laboratory growth conditions. SD: standard deviation.

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It has been suggested that grana stacking plays an important role in protecting photosystem II (PSII; located to the stacked grana) under high light irradiance (Anderson and Aro, 1994). To test this hypothesis, we examined the stress responses of ga1-3 and wild-type plants exposed to high light irradiance (Appendix S2). During the high light treatment, the decline of the Fv/Fm ratio, which reflects the maximum potential capacity of the PSII photochemical reactions (Krause and Weis, 1991), was more significant in the wild-type plants than in ga1-3 (Figure 5b), whereas during the recovery stage, the recovery of the Fv/Fm ratio was faster in ga1-3 than in the wild type. Our results suggested that ga1-3 is more resistant to oxidative damage than the wild type, and that this correlates with an increase in chloroplast grana stacking.

GA regulates chloroplast division and development through DELLA repressors

Arabidopsis has five DELLA proteins that repress GA responses: RGA, GAI, RGL1, RGL2 and RGL3 (Feng et al., 2008). To investigate their roles in GA-regulated chloroplast division and development, we chose gai-1 (a GAI semi-dominant mutation that confers GA-insensitive dwarfism, and contains a stable version of the GAI protein), rga-24 gai-t6 (null alleles of AtGAI and AtRGA) and della (a pentuple mutant of all five DELLA genes) for this study (Figure 6a). A significant decrease (by 10.3%) in the number of chloroplasts per mesophyll cell was found in the gai-1 mutant (Figure 6b), corresponding with a significant decrease in chloroplast division frequency (by 40.9%) and the chloroplast number per leaf (by 47.4%) in the gai-1 mutant, compared with the wild type (Figure 6c,d), suggesting that GAI negatively regulates chloroplast division. Consistent with this notion, the transcript levels of chloroplast division genes AtPDV1, AtPDV2, AtARC5 and AtFtsZ2-1 were reduced in gai-1 (Figure 6e). In contrast, an increase in chloroplast division frequency (by 48.1%) and chloroplast number per leaf (by 23.6%) was observed in the rga-24 gai-t6 mutant (Figure 6c,d), suggesting that RGA and GAI are the major repressors regulating GA-induced chloroplast division in Arabidopsis. However, in the pentuple mutant della, the chloroplast division phenotype is comparable with that of wild-type plants (Figure 6b–e). Quantitative RT-PCR analyses showed that the ARC5 level, but not those of other chloroplast division genes, was significantly increased in rga-24 gai-t6 (Figure 6e). However, the transcriptional levels of all the selected chloroplast division genes in the della mutant were comparable with those of wild-type plants (Figure 6e). Two possible reasons could explain the results described above: firstly, the effect of the rga-24 and gai-t6 mutations are antagonized by the loss of RGL1, RGL2 and RGL3 function; secondly, under DELLA-depleted conditions (such as in della mutant), additional unidentified factors are likely to be involved in maintaining chloroplast division, thereby preventing the formation of an excessive number of chloroplasts in the mesophyll cells.

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Figure 6.  DELLAs are also involved in the regulation of chloroplast division in Arabidopsis. (a) Approximately 3-week-old gai-1, rga-24 gai-t6 and della mutants and wild-type plants (scale bar: 1 cm). (b) Chloroplasts in the isolated mesophyll cells from the center of the fourth leaf of gai-1, rga-24 gai-t6 and della mutants and wild-type plants (scale bar: 10 μm). Numbers indicate the average chloroplast numbers per mesophyll cell (= 39 cells, ±SD). *Statistically significant differences, assessed by Student’s t-tests (< 0.05). SD, standard deviation. (c) Statistical comparison of the chloroplast division frequency in the fourth leaf tip (n ≥ 350 chloroplasts, ±SD). *Statistically significant differences, assessed by Student’s t-tests (< 0.05). (d) Comparison of the number of chloroplasts per leaf (fourth leaf, = 3 leaves). (e) Transcript levels of selected chloroplast-dividing genes in the fifth leaf were determined by quantitative RT-PCR. The error bars represent SDs.

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To further investigate the roles of DELLA proteins in the regulation of chloroplast division, we examined the chloroplast division phenotypes in rga-24 gai-t6 and della mutants in the presence of the GA biosynthesis inhibitor paclobutrazol (PAC). The chloroplast division frequency of rga-24 gai-t6 (with the RGL1, RGL2 and RGL3 still present) was partially inhibited by PAC (Figure 6c,d); in addition, quantitative RT-PCR analyses showed that the transcript levels of ARC5 in rga-24 gai-t6 can be partially inhibited by PAC (Figure 6e). These results suggest that RGL1, RGL2 and RGL3 also play a role in repressing chloroplast division. By contrast, the chloroplast division frequency of the della mutant showed a PAC-insensitive phenotype (Figure 6c,d), and the transcript levels of all the selected chloroplast division genes in the della mutant are comparable with those of the wild type (Figure 6e). These results confirm an essential role for all five DELLA proteins in the repression of chloroplast division, and indicate that additional unidentified factors are probably involved in maintaining chloroplast division under DELLA-depleted conditions.

To elucidate the role of DELLAs in the regulation of thylakoid network organization, we examined the chloroplast ultrastructure of gai-1, rga-24 gai-t6 and della mutant plants. An increased grana stacking was found in gai-1 (Figure 7a; Table S1), similar to changes observed in the ga1-3 mutants. On the contrary, rga-24 gai-t6 plants contained a lower grana stacking, displaying a grana-stacking phenotype similar to that of wild-type plants treated with exogenous GA (Figure 5a), and the application of PAC increased grana stacking in rga-24 gai-t6 (Figure 7b; Table S1). These observations suggest that RGA and GAI are the major regulators for GA-regulated grana stacking in Arabidopsis. In the della mutant, the grana stacking phenotype is comparable with that of wild-type plants, and PAC has no significant effect on grana stacking in the della mutant (Figure 7c; Table S1), suggesting that RGL1, RGL2 and RGL3 also play a role in regulating grana stacking.

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Figure 7.  DELLAs are also involved in the regulation of grana stacking in Arabidopsis. Ultrastructure of the chloroplasts in the top layers of mesophyll cells of gai-1 (a), rga-24 gai-t6 (b) and della (c) mutant plants, with or without the application of PAC. Scale bars: 1 μm (1, 3, 5, 7 and 9) and 0.17 μm (2, 4, 6, 8 and 10).

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Gibberellins are required for chloroplast biogenesis in Oryza sativa

To investigate whether GA regulates chloroplast biogenesis in monocotyledon plants, we examined whether GA plays a similar role in regulating chloroplast biogenesis in Oryza sativa (rice). We first examined the leaf morphology and anatomy of the third leaf blade of d18-AD (a loss-of-function mutant in the rice GA biosynthesis gene 3β-hydroxylase; Murakami, 1972) and wild-type plants (Figure 8a). A significant decrease in leaf area (by 52.6%; Figure 8a), corresponding with a decrease in cell number per leaf (by 55.6%; Figure 8b), was found in d18-AD when compared with the wild type, suggesting that GA plays a critical role in regulating cell division during leaf growth and development in rice. We next examined the leaf anatomy of the base and tip portions of the third leaf blade of d18-AD and wild-type plants. The base portion represents an earlier developmental stage than the tip portion. An increase in chloroplast number per unit leaf area (by 30.1% in leaf base and by 25.1% in leaf tip) was found in the cross-sections of d18-AD, compared with the wild type (Figure 8c), corresponding with an increase in chlorophyll content per unit leaf area (by 49.7%; Figure 8d) and a higher photosynthesis rate in d18-AD, compared with the wild type (Figure 8e). These observations suggest that the increased pigmentation in the d18-AD mutant can be attributed to an increase in chloroplast number per unit leaf area, which is similar to the dark-green leaf phenotype of ga1-3.

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Figure 8.  Gibberellin (GA) regulates chloroplast division in Oryza sativa (rice). (a) Two-week-old wild-type and d18-AD mutants (scale bar: 0.5 cm). The arrows indicate the third leaves, which were used for the experiments. Numbers indicate the average leaf area (= 3 leaves, ±SD). SD, standard deviation. (b) Comparison of the cell number per leaf of the wild type and d18-AD mutant (= 3 leaves, ±SD). (c) Cross sections of the third leaves (base and tip) of wild-type and d18-AD mutant plants (scale bar: 25 μm). Numbers indicate the average chloroplast numbers per unit leaf area (= 3 leaves, ±SD). (d) Comparison of the chlorophyll content per unit leaf area (1 cm2) in the wild type and d18-AD. The error bars represent SD (= 3 leaves, ±SD). (e) The oxygen evolution rate for unit leaf area (2 cm2) was measured under the light intensity of 690 μmol m−2 sec−1. A representative experiment is shown. Two biological replicates gave the same result. (f) Statistical comparison of the chloroplast division frequency in the leaf bases of the third leaf (≥ 116 chloroplasts). *Statistically significant differences, assessed by Student’s t-tests (< 0.05). (g) Comparison of the chloroplast number per leaf of the wild type and the d18-AD mutant (= 3 leaves, ±SD). (h) The transcript levels of rice homologs of chloroplast division genes in the elongating third leaf were determined by quantitative RT-PCR. The error bars represent SDs.

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We next examined the chloroplast division phenotype in d18-AD. A significant decrease in the chloroplast division frequency (by 61.2%) and chloroplast number per leaf (by 42.9%) was found in d18-AD (Figure 8f,g), suggesting that chloroplast division was suppressed in d18-AD. However, we found that d18-AD contained 37.6% more chloroplasts per mesophyll cell than the wild type (Figure S1), but that cell division was suppressed to a larger extent (Figure 8b). Together with the observation that the cell number per leaf was decreased by 55.6% (Figure 8b), whereas the chloroplast number per leaf was decreased by 42.9% (Figure 8f) in d18-AD, these results suggest that the increased chloroplast number per mesophyll cell in the d18-AD mutant may be caused by a more drastic decrease in cell division rate and a more moderate decrease in chloroplast division rate in the d18-AD mutant. Quantitative RT-PCR analysis showed that the transcript levels of the rice homologs of the Arabidopsis chloroplast division-related genes, including OsPDR1-1, OsARC3, OsPDV2 and OsPDV1-2, were decreased in the d18-AD mutant when compared with the wild type (Figure 8h). Furthermore, ultrastructural observations showed that d18-AD chloroplast had an increase of grana stacking compared with the wild type (Figure 9a,b; Table S2). Together, the results described above provide evidence that GAs play a similar role in the regulation of chloroplast biogenesis in monocotyledon and dicotyledon species.

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Figure 9.  Gibberellins (Gas) are involved in the regulation of grana stacking in Oryza sativa (rice). (a, b) Ultrastructure of the chloroplasts in the mesophyll cells of leaf tips and leaf bases in the third leaves of wild-type and d18-AD mutant plants. Scale bars: 2 μm (1, 4, 7, and 10), 1 μm (2, 5, 8, and 11) and 0.17 μm (3, 6, 9, and 12).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The increased chloroplast density causes the dark-green rosette phenotypes in GA-deficient mutants

Our study found that the Arabidopsis GA-deficient mutant ga1-3 displays more tightly packed mesophyll cells and thicker mesophyll cell layers when compared with the wild-type leaves (Figure 1b), suggesting that GA plays an important role in leaf development by regulating mesophyll cell arrangement. Furthermore, we found that the ga1-3 and d18-AD mutants display an increase in chloroplast number per unit leaf area (Figures 1f and 8c), consistent with an increase in chlorophyll content per unit leaf area (Figures 1g and 8d). Thus, our data suggested that the dark-green rosette phenotype of GA-deficient mutants is caused by an increase in chloroplast density and chlorophyll content per unit leaf area within the leaves.

Moreover, an increase of chloroplast density per cell was found in ga1-3 (Figure 2c), corresponding with a steeper slope of the best-fitting line for the chloroplast numbers per cell versus the cell area in ga1-3 than in the wild type (Figure 2b). Notably, it has been suggested that the smaller slopes of the best-fitting line indicate the more severe chloroplast division deficiency (Pyke and Leech, 1992; Schmitz et al., 2009). For example, the chloroplast division mutants arc2 and arc3 showed significant reductions in the number of chloroplasts per cell, corresponding with a smaller slope of the best-fitting line for the chloroplast numbers per cell versus the cell area, when compared with the wild type (Pyke and Leech, 1992). In this study, we found a decreased chloroplast number per leaf (Figure 3a), consistent with a decreased chloroplast division frequency in the ga1-3 mutant (Figure 3b), suggesting that chloroplast division deficiency may also occur in mutants with steeper slopes. Taken together, the data described above suggest that the severity of the block in chloroplast division should be determined not only at the cellular level but also at the organ level.

GA coordinately regulates chloroplast division, cell expansion and cell division

In this study we showed that chloroplast number per leaf was significantly reduced in the severe GA-deficient mutants ga1-3 and d18-AD (Figures 3a and 8g), accompanied by a reduction of the transcript levels of chloroplast division genes (Figures 3c and 8h), suggesting that GAs plays a positive role in the regulation of chloroplast division and chloroplast division-related gene expression. Moreover, we found that the decrease of chloroplast number per leaf (by 72.8%) in ga1-3 (Figure 3a) was in parallel with the decrease of cell number per leaf (by 75.1%) in ga1-3 (Figure 1e), suggesting that chloroplast division and cell division are highly coordinated. We speculated that GA may indirectly regulate chloroplast division through its regulation of cell expansion and/or cell division, based on the following evidences. Firstly, high rates of plastid division are usually found in the tissues in which cell expansion and division are rapid, such as at the base of Arabidopsis petals and in a young Triticum spp. (wheat) leaf. Secondly, artificially increasing cell size results in increased chloroplast divisions (Pyke, 1997). Thirdly, a time-course analysis of the effects of exogenous GA treatment on the growth of ga1-3 leaves showed that the cell expansion-related genes tended to be upregulated much earlier, and more significantly, than the genes related to chloroplast division and cell division (Figures 4c,e,g). These data suggest that GAs may indirectly promote chloroplast division through their impact on leaf mesophyll cell expansion. This notion is also consistent with a previous microarray study of gene expression during Arabidopsis seed germination (Ogawa et al., 2003).

GA regulates chloroplast division through DELLA proteins

In this study, we found a significant increase in chloroplast division frequency and chloroplast number per leaf in rga-24 gai-t6, when compared with the wild type; however, the chloroplast division phenotype of the pentuple mutant della is comparable with that of wild-type plants (Figure 6c,d). Furthermore, the chloroplast division frequency of rga-24 gai-t6 was partially inhibited by PAC, whereas the chloroplast division frequency of the della mutant showed a PAC-insensitive phenotype (Figure 6c,d). Together with the observation that the level of GAI and RGA message is high in rosette leaves, whereas the other DELLA subfamily members (RGL1, RGL2 and RGL3) have weak or undetectable expression in rosette leaves (Wen and Chang, 2002), these results suggest that RGA and GAI are the major repressors in regulating GA-induced chloroplast division, but other DELLA proteins also play a role in repressing chloroplast division. Moreover, additional unidentified factors are likely to be involved in maintaining chloroplast division under DELLA-depleted conditions. It has been reported that, SPATULA (SPT), a phytochrome-interacting factor (PIF) homolog, acts in an analogous manner to DELLAs, which restrain cotyledon expansion alongside SPT (Josse et al., 2011). Cross-regulation of SPT by DELLAs ensures that the levels of SPT and DELLAs are tightly coordinated to prevent excessive or deficient growth. Under DELLA-depleted conditions, SPT plays a role in maintaining growth restraint. Thus, it will be interesting to investigate whether SPT is involved in the control of chloroplast division, and how it coordinately regulates chloroplast division within the GA signaling pathway.

Recent studies suggest that DELLA proteins are transcriptional regulators. However, DELLA proteins do not have a clearly identified DNA-binding domain, and they may associate with their target promoters by interacting with other transcription factors (Zentella et al., 2007). Consistent with this, it was recently shown that in the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 and 4 (PIF3 and PIF4), and prevent PIF3 and PIF4 from binding to their target gene promoters and regulating gene expression, and therefore abrogate PIF3- and PIF4-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) directly interact with DELLA proteins in the nucleus, trigger DELLA protein degradation and thus release PIF proteins from the negative effect of DELLA proteins (Feng et al., 2008; de Lucas et al., 2008). Chromatin immunoprecipitation (ChIP) and microarray data indicate that some PIF proteins (e.g. PIF1) do not bind or regulate the chloroplast (plastid) division-regulated genes during seed germination. Instead, PIFs could directly bind to the promoter regions of several cell division and expansion-related genes (one cyclin-dependent kinase gene, CDKD1;1; three cytokinin signaling genes, CRF2, CRF3 and CRF4; two expansin genes, EXP8 and EXP10; and one xyloglucan endotransglyco-sylase homolog, XTH28; Oh et al., 2009). In addition, PIF1 also indirectly represses the expression of four other EXP genes and six other XTH genes (Oh et al., 2009). These results demonstrate a close interplay between light and GAs in regulating cell expansion/elongation, and subsequently impacting on chloroplast division.

A new compensatory system of photosynthetic membrane, linking chloroplast number and grana stacking

Genetic evidence shows that a compensatory system(s) is involved in leaf morphogenesis. For example, the reduction in the final leaf area is compensated by an increase in the size of the leaf cells. Similarly, an increase in cell volume can trigger a decrease in cell number, and vice versa (Tsukaya, 2003; Ferjani et al., 2007). At the organelle level, the reduction in the chloroplast number is compensated by an increase in the size of chloroplast (Pyke and Leech, 1992). However, the compensatory phenotypes described above were not found in our study: instead, several new compensatory system(s) involved in leaf morphogenesis were found in response to GA deficiency. Firstly, the reduction of the final leaf area (Figures 1a and 8a) was compensated by an increase of chloroplast number per unit leaf area in the ga1-3 and d18-AD mutants (Figures 1f and 8c), which corresponds with an increased chlorophyll content per unit leaf area (Figures 1g and 8d) and an increased rate of photosynthesis (Figures 1h and 8e). Secondly, the reduction of the chloroplast number per leaf (Figures 3a and 8g) was compensated by an increase of grana stacking in the ga1-3 and d18-AD mutants (Figures 5a and 9a,b). Our results suggest that compensation reflects an organ-wide coordination of chloroplast density and thylakoid membrane arrangement in determinate organs, which may be necessary for the GA-deficient mutants to modulate the proper photosynthesis rate in response to decreased leaf area. Similar compensatory pathways were found in Arabidopsis and rice in response to GA deficiency.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Leaf anatomy and chloroplast morphology

The leaf center of Arabidopsis expanding leaves (fourth leaf of 3-week-old plants), the base (one-quarter) and tip (one-quarter) portions of rice elongating leaves (third leaf of 2-week-old plants) were collected at approximately 10 00 h, and fixed in 4% (v/v) glutaraldehyde in 0.1 m phosphate buffer solution (pH 7.2) for 4 h at 4°C, and then post-fixed in 1% OsO4 for 1 h at 25°C. After dehydration through an acetone series, the samples were embedded in Epon812 and subsequently polymerized.

For the light microscope observation, sections (1–2 μm in thickness) were stained with Toluidine Blue O and were photographed using a Leica DMLB light microscope Leica DFC320 camera. The chloroplast number per unit leaf area was counted from the cross sections (550 μm in width of the fourth leaf for Arabidopsis; 275 μm in width of the third leaf for rice).

For the ultrastructural observations, ultrathin sections (50–70 nm in thickness) were stained with uranyl acetate (2.5%, w/v) and lead citrate. Chloroplasts in the most upper layer of mesophyll cells were observed and photographed using a Philips FEI-Technai 12 microscope, http://www.fei.com/. The number of stacked thylakoid membranes per granum was counted from 40 to 50 stochastic grana in the chloroplasts (≥ 3 chloroplasts).

For observing chloroplasts in isolated cells from Arabidopsis, the leaf centers and leaf tips of the fourth leaves were fixed with 3.5% glutaraldehyde at 4°C overnight, and then incubated in 0.1 m Na2-EDTA, pH 9.0, for 15 min at 50°C (Okazaki et al., 2009). The isolated mesophyll cells were observed with Nomarski optics (Leica, http://www.leica.com). The area of the fully expanded palisade mesophyll cells at the leaf center (≥ 30 cells) was measured using ImageJ. The chloroplast number per corresponding mesophyll cell was counted. The chloroplast division frequency (measured as the percentage of dumbbell-shaped chloroplasts per random selected chloroplasts, n ≥ 350) was counted from the isolated mesophyll cells from the leaf tip.

For the observation of the chloroplasts in rice isolated cells, the base and center portions from the third leaves of rice were fixed with 3.5% glutaraldehyde at 4°C overnight, and then incubated in 0.1 m Na2-EDTA, pH 9.0, for 1 h at 50°C. The chloroplast division frequency was counted from the chloroplasts (≥ 116 chloroplasts) of the leaf base. The chloroplast number in the fully expanded mesophyll cells (= 30) was counted from the leaf center.

Estimation of chloroplast number and cell number per leaf

Based on the linear relationship between the chlorophyll content per unit leaf area, the chlorophyll content of a single chloroplast and the chloroplast number per unit leaf area (Karia and Tsunoda, 1972), we estimated the chloroplast number per single leaf by dividing the chlorophyll content per single leaf (μg) by the chlorophyll content per single chloroplast (μg). The intact chloroplasts were isolated using a Chloroplast Isolation Kit (CP-ISO; Sigma-Aldrich, http://www.sigmaaldrich.com), according to the manufacturer’s instructions. To obtain the chlorophyll content per single chloroplast, the weight (μg) of the chlorophyll per microlitre of the chloroplast suspension was divided by the chloroplast number per microlitre of the chloroplast suspension.

For Arabidopsis, we estimated the cell number per leaf by dividing the average leaf area by the average mesophyll cell plan area, and then multiplied the average by the mesophyll cell layer number that was counted in the cross section. For rice, we estimate the cell number per leaf by multiplying the cell number per unit leaf area (counted from the cross section of the middle of the leaf) by the leaf area.

Kinematic analysis of cell expansion and chloroplast division in response to GA treatment

On the day of the experiment, 2-week-old ga1-3 seedlings were sprayed with either 100 μm GA3 or water (control) every 30 min, starting from 10 00 h, for the entire 48 h of the experiment, and true leaves were collected from seedlings at 2, 24 and 48 h after 10 00 h. The measurement of chloroplast number per cell, chloroplast division frequency, cell division rate (average cell division rates for the whole leaf were determined as the slope of the log 2-transformed number of cells per leaf; De Veylder et al., 2001) and mesophyll cell area was performed on the first true leaves. For quantitative RT-PCR analyses, total RNA was extracted from all of the true leaves. Kinematic analyses were performed on two experimental repeats.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Matthew J. Terry (University of Southampton), Jitao Zou (National Research Council, Canada), Chanhong Kim (Boyce Thompson Institute for Plant Research) and Ling Yuan (University of Kentucky) for helpful discussions, Xiangdong Fu (Institute of Genetics and Developmental Biology of Chinese Academy of Sciences) for providing us with the gai-1, rga-24 gai-t6 and della seeds and Qian Qian (China National Rice Research Institute) for providing us with the d18-AD seeds. This study was supported by the Nature Science Foundation of China (project no. 90917011) to XW and (project no. 31070159) to HW and (project no. 31200151) to XJ.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Chloroplasts in mesophyll cells inrice. (a) Chloroplasts in isolated mesophyll cells from the centreof the third leaf observed by Nomarski optics (Bar=10 μm).(b) Statistical comparison of the number of chloroplasts permesophyll cell (= 30 cells) from the third leaf.Error bars represent SD. Asterisk indicates statisticallysignificant differences assessed by t-tests(< 0.01). SD, standard deviations.

Table S1. The number of thylakoid membranelayers per granal stack in chloroplasts of Arabidopsis ga1-3,gai-1, rga-24/gait-6 mutants and the wild-type, with or without the treatment of GA or PAC.

Table S2. The number of thylakoid membranelayers per granal stack in the chloroplasts from the leaf base andleaf tip of rice d18-AD mutant and the wild-type.

Table S3. Primers used in this study.

Appendix S1. Supplemental experimental procedures.

Appendix S2. The chlorophyll content (lg) per chloroplast in Arabidopsis ga1-3 mutant and the wild-type, with or without the treatment of GA..

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