Introduction

Leaves are the main photosynthetic organ in plants. In addition, the number and arrangement of leaves greatly contributes to the establishment of plant shape. To understand the genetic mechanism underlying shoot formation, two aspects of leaf primordial formation must be considered: spatial (phyllotaxy) and temporal (plastochron) regulation. Given that the regular phyllotactic pattern has long fascinated plant scientists, a large number of studies have been conducted to investigate this phenomenon (for review, Steeves and Sussex 1989). However, rapid progress has been achieved only recently. In maize, the causal gene of the abphyl1 mutant, which exhibits decussate phyllotaxy instead of 1/2 alternate, was isolated in 2004 (Giulini et al. 2004). The recent auxin-based model has been widely accepted (Reinhardt et al. 2000, 2003; Jönsson et al. 2006; Smith et al. 2006).

In contrast, the molecular basis of plastochron regulation remains to be uncovered. plastochron 1 (pla1) is the first mutant that drastically alters plastochron, in which leaf primordia are formed approximately twofold faster than in the wild type (Itoh et al. 1998). Concomitantly, leaves of pla1 become short, suggesting that PLA1 regulates organ size. The PLA1 gene encodes a cytochrome P450 family protein (CYP78A11) (Miyoshi et al. 2004), but its substrate is unknown. An Arabidopsis homolog of PLA1, KLUH, was shown to regulate organ size (Anastasiou et al. 2007). Subsequently, the PLA2 and PLA3 genes, loss-of-function mutants of which exhibit similar phenotypes to that of pla1, were identified (Kawakatsu et al. 2006, 2009). These encode an RNA-binding protein and glutamate carboxypeptidase, respectively. Interestingly, PLA1 and PLA2 are expressed in young leaf primordia, but not in shoot meristems (Miyoshi et al. 2004; Kawakatsu et al. 2006). Therefore, based on analyses of the developmental processes of leaves, the primary functions of PLA1 and PLA2 are suppression of precocious leaf maturation, and that some non-cell autonomous signals move from leaf primordia through shoot meristems to suppress the formation of a new leaf primordium (Kawakatsu et al. 2006). Thus, PLA1 and PLA2 are key genes for elucidating leaf development. However, the regulation of PLA1 and PLA2 expression remains unknown.

pla1 and pla2 show several phenotypes likely related to phytohormones, such as small leaf size, dwarfism and enlarged SAM. In addition, the phytohormone (CK, abscisic acid and IAA) contents of pla mutants differed from those of the wild type (Kawakatsu et al. 2009). These mutant phenotypes suggest that PLA genes have some relationship with phytohormones. However, the position of PLA genes in the phytohormone-related pathway remains unclear.

Several phytohormones are involved in the regulation of leaf development/growth. Auxin has pleiotropic functions on plant development, including leaf growth (for review, Teale et al. 2006). Rice tryptophan deficient dwarf 1 (tdd1) mutant exhibits low auxin content and dwarfism with small leaves (Sazuka et al. 2009). A gain-of-function mutant of rice, OsIAA3, which inhibits auxin signaling, shows an auxin-insensitive phenotype, and produces shorter leaves than the wild type, resulting in dwarfism (Nakamura et al. 2006a). Thus, auxin biosynthesis and signaling is important for normal leaf development and morphological processes in rice. Cytokinine (CK) is also profoundly associated with leaf development. For example, ABPYL1, which encodes the A-type response regulator, regulates phyllotaxy in maize (Jackson and Hake 1999; Giulini et al. 2004). Overexpression of a type-A response regulator caused dwarfism in rice (Hirose et al. 2007). Another phytohormone, brassinosteroid (BR), has a role in regulating plant growth. Loss-of-function mutants of BR biosynthetic and signaling genes frequently exhibit dwarfism and a reduced organ size. Rice D2 and D11 are BR biosynthesis genes, and regulate grain size and other traits (Hong et al. 2003; Tanabe et al. 2005). A BR-insensitive mutant, d61, also exhibits dwarfism, and a severe d61 allele, d61-4, exhibited rolled and twisted leaves (Yamamuro et al. 2000; Nakamura et al. 2006b).

Gibberellin (GA) is the most well-known phytohormone that affects plant height and organ (leaf) size. Semi-dwarf GA mutants were used in the wheat and rice green revolution (Hedden 2003). In rice, many dwarf mutants are associated with GA biosynthesis or signaling. For example, SEMIDWARF 1 (SD1), which encodes GA20 oxidase, was utilized in the rice green revolution (Ashikari et al. 2002; Sasaki et al. 2002). The d18 dwarf mutant of GA3ox2 has an extremely dwarf stature with small leaves (Itoh et al. 2001), and a prolonged juvenile phase (Tanaka et al. 2011). Other GA-deficient and GA-insensitive mutants commonly exhibit small leaves and a dwarf stature. In contrast, GA promotes the juvenile-adult phase change (Evans and Poehtig 1995; Teifer et al. 1997; Schwartz et al. 2008). In wild-type rice, plastochron is short in the juvenile compared with the adult phase (Itoh et al. 2005). Thus, GA seems to be associated with PLA functions, whose mutants show a short plastochron.

We examined the sensitivities of pla1 and pla2 to several phytohormones, and revealed that GA is the major influencer of PLA function. Using GA-related genes and mutants thereof, we determined that PLA1 and PLA2 function downstream of GA signal transduction.

Materials and methods

Plant materials

We used pla1-4 and pla2-1 mutants, which show the most severe phenotypes among their alleles, and share a cv Taichung 65 genetic background (Kawakatsu et al. 2006). We also used a GA-biosynthetic dwarf mutant, d18-h, which encodes GA3 oxidase2 and has a low auxin content (Itoh et al. 2001). Three GA signaling mutants, gibberellin insensitive dwarf 1 (gid1), slender rice 1-1 (slr1-1) and Slr1-d1 were used (Ikeda et al. 2001; Asano et al. 2009). GID1 encodes a GA receptor, thus its loss-of-function mutant is GA insensitive (Ueguchi-Tanaka et al. 2005). SLR1 encodes the DELLA protein and plays an important role in GA signal transduction (Ikeda et al. 2001; Itoh et al. 2002; Gomi et al. 2004). slr1-1 forms a highly-elongated plant due to constitutive activation of GA signaling, while Slr1-d1 shows dwarf phenotype and is a dominant allele of SLR1 (Ikeda et al. 2001; Asano et al. 2009).

Application of phytohormones

Wild-type and mutant seeds were sterilized with 1 % NaClO for 40 min, and washed four times in sterile distilled water. The seeds were then placed on the Murashige and Skoog (1962) medium containing various concentrations of 2,4-D, kinetin, GA3 24-epiBL or uniconazole. Plants were grown in a growth chamber under continuous light at 28 °C. After 10 or 14 days, plant height and second leaf sheath length were measured for more than five plants of each treatment.

Clearing of leaf sheath and measurement of cell size

To measure cell size, leaf sheaths were fixed with FAA (formalin: acetic acid: 50 % ethanol, 1:1:18) for 24 h at 4 °C. They were then dehydrated in a graded ethanol series and cleared in chloral hydrate at 96 °C in a heat block. We measured epidermal cell sizes on the adaxial side of cleared leaf sheaths under a light microscope. Measurements were performed on at least 100 cells per sample. Significant differences were analyzed by Student’s t test.

In situ hybridization

Ten-day-old shoot apices of wild-type plants treated with GA3 or uniconazole and of slr1-1 plants were fixed with paraformaldehyde in 0.1 M sodium phosphate buffer, dehydrated through a series of butanol extractions, and embedded in paraplast plus. Microtome sections (8 mm thick) were applied to glass slides coated with APS (Matsunami Glasses, Japan). A digoxigenin-labeled antisense probe of PLA1 was prepared as described previously (Miyoshi et al. 2004). Hybridization and immunological detection with alkaline phosphatase were preformed as described by Kouchi and Hata (1993).

Real-time PCR

Total RNA was extracted from shoot apices using TRIZOL reagent (Invitrogen). After RNase-free DNase I treatment, 1 μg of RNA was used for RT-PCR using High-capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA). To quantify PLA1, PLA2, GA20ox2, GA3ox2, GA2ox4 and SLR1 expression, real-time PCR was performed using SYBERGREEN (Applied Biosystems, USA) or the TaqMan Fast Universal PCR Master Mix, FAM-labeled TaqMan probes (Applied Biosystems, USA), and the StepOnePlus real-time PCR system (Applied Biosystems, USA). Each gene expression value is the average of three independent real-time PCR assays. Expression levels were normalized to that of an internal control, ACT1. The primers and probes for each gene are listed in Supplementary Table S1.

Results

Since the pla1 and pla2 mutants exhibit dwarf phenotypes and short plastochron (rapid leaf emergence), it is hypothesized that pla phenotypes are related to phyotohormones. Of the many pla1 and pla2 alleles, we used pla1-4 and pla2-1, both of which are strong alleles with a common genetic background of cv. Taichung 65.

Gibberellin is the major phytohormone associated with PLA1 and PLA2 functions

First, we observed the responses of pla1 and pla2 seedlings when several phytohormones, which are known to affect plant growth and leaf development, were added to the culture media. Both pla1-4 and pla2-1 mutants showed similar responses to auxin (2,4-D) as did the wild type. That is, 2,4-D inhibited the growth of wild-type, pla1-4 and pla2-1 seedlings (Fig. 1a). CK (kinetin) application caused similar responses in wild-type and pla seedlings (Fig. 1b). Higher kinetin concentrations caused more severe growth inhibition in wild-type, pla1-4 and pla2-1 seedlings. BR is also known to affect plant height and leaf development. However, both pla1-4 and pla2-1 seedlings responded to external BR (24-epiBL) similarly to wild-type seedlings (Fig. 1c). Thus auxin, CK and BR are not related to PLA function. In contrast, GA3 application induced rapid growth of wild-type plants (Fig. 1d, e). In pla1-4 and pla2-1, however, growth induction was restricted (Fig. 1d, e). Accordingly, GA is the major phytohormone associated with PLA1 and PLA2 functions, while pla1-4 and pla2-1 seem to be less sensitive to GA than wild-type plants.

Fig. 1
figure 1

Effect of several phytohormones on the growth of pla1 and pla2 seedlings. Wild-type, pla1-4 and pla2-1 seeds were inoculated on culture media containing phytohormones. Plants were grown for 14 days in 2,4-D (a) and kinetin (b) treatments, and for 10 days in 24-epiBL (BR) (c) and GA3 (d) treatments. Data in a–d represent mean ± SE. e Seedlings of wild type, pla1-4 and pla2-1 grown for 10 days with or without GA3 treatment. Bar 2 cm

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Responses of pla1 and pla2 to gibberellin application in cell size and leaf elongation

We examined responses to GA in cells and tissues of pla1-4 and pla2-1 plants. GA promotes cell elongation and tissue/organ elongation. We measured the length of more than 100 cells on the adaxial surface of the second leaf sheath. In wild-type plants, GA3 application elongated leaf sheath cells by approximately 18 %, but by at most 6 % in pla1-4, and no elongation was observed in pla2-1 plants (Fig. 2a, b). Leaf sheaths elongate in response to GA3 application. In wild-type plants, elongation of the 2nd leaf sheath increased with GA3 concentration, being circa threefold longer at 10−5 than at 0 M, whereas pla1-4 and pla2-1 showed a twofold or less elongation at the same GA3 concentrations (Fig. 2c). In particular, a low GA3 concentration did not promote pla1-4 and pla2-1 leaf elongation.

Fig. 2
figure 2

Response of pla1 and pla2 to gibberellin application. a Adaxial surface of cleared 2nd leaf sheath of wild type, pla1-4 and pla2-1 treated with GA3. Bars 200 μm. b Effects of GA3 treatment on cell length in 2nd leaf sheath. In WT, GA3 treatment significantly increased cell length. Double asterisks significantly longer at 1 % level than in −GA (t test). c Effects of GA3 treatment on the length of 2nd leaf sheath. Double asterisks significant at 1 % level compared with 0 M (t test). Data in b, c represent mean ± SE

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These results indicate that pla1-4 and pla2-1 are insensitive to GA in many traits, suggesting that PLA1 and PLA2 act downstream of GA signal transduction.

Induction of PLA gene expression by gibberellin

The above results suggest that PLA1 and PLA2 gene expression is associated with GA signaling. Thus, we examined the effect of GA on PLA1 and PLA2 gene expression. Ten-day-old seedlings were treated with 10 μM GA3 and PLA gene expression was monitored for 24 h by real-time PCR. PLA1 and PLA2 expression increased as early as 3 h after treatment, and a high level of expression was maintained for 24 h (Fig. 3a). To investigate the long-term effect of GA, wild-type seeds were inoculated and grown for 8 days on culture media containing 10 μM GA3. PLA1 and PLA2 expression was maintained at a high level for 8 days (Fig. 3b). Next, we examined the effect of uniconazole, a GA biosynthesis inhibitor. Uniconazole treatment markedly suppressed PLA1 and PLA2 gene expression (Fig. 3b). Therefore, GA regulates the expressions of both PLA1 and PLA2.

Fig. 3
figure 3

Induction of PLA1 and PLA2 expression by gibberellin. a, b Real-time PCR assays. a Short-term induction of PLA1 and PLA2 expression by 10 μM GA3. Each expression level is represented relative to that at 0 h. b Effects of GA3 and uniconazol treatments for 8 days on PLA 1 and PLA2 expressions. Expression level is represented relative to that in the control. Data in a, b represent mean ± SE. c–e In situ hybridization of PLA1 in wild-type shoot apex (c), wild-type shoot apex treated with 10 μM GA3 for eight days (d) and wild-type shoot apex treated with 1 μM uniconazole for 8 days (e). Arrows indicate ectopic expression. Bars 100 μm

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Since GA and uniconazole treatments up- and down-regulated PLA1 expression, respectively, the PLA1 expression pattern was investigated using in situ hybridization. PLA1 is expressed in the basal and abaxial regions of leaf primordia, but not in shoot meristems (Fig. 3c, Miyoshi et al. 2004). When treated with GA3, strong and ectopic PLA1 expression was detected in the adaxial region of leaf primordia and in the tip of shoot meristems in addition to the normal expression domain (Fig. 3d). The expression pattern was not unaffected by uniconazole treatment, but hybridization signals were weakened (Fig. 3e).

Expression of GA-related genes in pla mutants

Given that GA regulates PLA1 and PLA2 expression, we next examined the expression of GA-related genes in pla1 and pla2 mutants. The expression of the GA biosynthesis genes, GA20ox2 and GA3ox2, and the GA-catabolizing gene, GA2ox4, did not largely differ among wild-type, pla1-4 and pla2-1 plants, although GA3ox2 expression was somewhat increased in pla1-4 and pla2-1 compared with the wild type (Fig. 4a). Similarly, SLR1 (a gene involved in GA signal transduction) expression was comparable in wild-type, pla1-4 and pla2-1 plants (Fig. 4a). These data suggest that PLA1 and PLA2 do not affect GA biosynthesis or signal transduction.

Fig. 4
figure 4

Expression of gibberellin-related genes in pla1 and pla2 seedlings. a–c Real-time PCR assays. a Expression of GA-biosynthetic (GA20ox2 and GA3ox2), catabolizing (GA2ox4) and signaling gene (SLR1) genes in wild type, pla1-4 and pla2-1. Expression level in pla mutants is represented relative to that in wild type. b, c Effect of GA3 and uniconazole treatments on GA20ox2 (b) and GA2ox4 (c) expression in wild type, pla1-4 and pla2-1. Each expression level is represented relative to that in wild-type control. In b and c, expression level of GA20ox2/GA2ox4 in the control (non-treatment) does not significantly differ among wild type, pla1-4 and pla2-1. Data in a–c represent mean ± SE

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In plants, GA content is regulated by a feedback mechanism involving GA signal transduction; e.g., GA20ox2 expression is enhanced in GA-insensitive mutants such as gibberellin insensitive dwarf 1 (gid1), and downregulated in the GA-constitutive-active mutant slr1-1 (Yamaguchi 2008). Therefore, we investigated whether GA signal transduction is operating normally in pla1 and pla2 by determining the effect of GA and an inhibitor thereof on expression of the above genes. Application of GA slightly decreased the expression of GA20ox2 in wild-type, pla1-4 and pla2-1 plants (Fig. 4b). In contrast, uniconazole treatment markedly enhanced expression in wild type and pla1-4, and moderately enhanced it in pla2-1, plants (Fig. 4b). The opposite effect was detected for GA2ox4, which encodes a GA-catabolizing enzyme. GA treatment strongly enhanced GA2ox4 expression in wild-type and pla1-4 plants (Fig. 4c). In pla2-1 plants, GA also induced the expression, but to a limited extent (Fig. 4c). Uniconazole treatment suppressed GA2ox4 expression in wild-type, pla1-4 and pla2-1 plants (Fig. 4c).

These results show that the feedback mechanism is operating normally in pla1-4 and pla2-1 mutants, although somewhat weakened in pla2-1. In addition, PLA1 and PLA2 may be positioned downstream of GA biosynthesis and signal transduction genes.

PLA gene expression in GA-related mutants

To confirm the relationship between PLA genes and GA-related genes, we examined PLA1 and PLA2 expression in GA-related mutants. d18 is a dwarf mutant, whose wild-type gene encodes GA3 oxidase 2. PLA1 and PLA2 expression in d18 was slightly down-regulated compared with the wild type (Fig. 5a). However, this difference in PLA expression between the wild type and d18-h was not large compared with that between the wild-type and GA-signaling mutants below. In GA-insensitive mutant gid1 and reduced GA-sensitivity mutant Slr1-d1, PLA1 and PLA2 expression was severely suppressed, whereas in the GA-constitutive-active mutant slr1-1, their expression was markedly increased (Fig. 5b).

Fig. 5
figure 5

PLA1 and PLA2 gene expression in GA-related mutants. a, b Real-time PCR assays of PLA1 and PLA2 expression in GA-deficient mutant (d18-h) (a) and in inactive (gid1 and Slr1-d1) and constitutive active (slr1-1) mutants of GA signaling (b). Expression level in each mutant is represented relative to that in wild type. Data in a, b represent mean ± SE. c–e in situ hybridization of PLA1 in wild-type shoot apex (c) and slr1-1 shoot apex (d, e). Arrows indicate ectopic expression. Bars 100 μm

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To determine the effect of PLA1 overexpression on the expression domain, we examined the PLA1 expression pattern by in situ hybridization. Compared with that in the wild-type shoot apex, PLA1 expression in slr1-1 was expanded to the adaxial region and the upper regions of leaf primordia and shoot meristems, as well as the normal basal and abaxial regions of leaf primordia (Fig. 5c–e). This ectopic expression of PLA1 coincided with that induced by GA treatment (Fig. 3d). Since PLA1 and PLA2 expression was strongly affected by GA-signaling genes, both PLA1 and PLA2 likely act downstream of the GA signal transduction pathway to regulate leaf development.

Discussion

GA is associated with various developmental and physiological processes, such as seed germination, stem elongation, vegetative phase change, flowering and pollen maturation (Olszewski et al. 2002; Yamaguchi 2008). It is also proposed that GA functions in early leaf development (Olszewski et al. 2002; Yamaguchi 2008). A subset of class I KNOX genes represses GA-biosynthetic gene expression in the SAM by direct transcriptional regulation, resulting in prevention of SAM cells from entering a determinate state (Sakamoto et al. 2001) Once a leaf is initiated from the shoot apex, negative regulation of GA occurs in the leaf primordium, and GA contributes to leaf expansion and differentiation (Olszewski et al. 2002; Yamaguchi 2008). This model is widely accepted, and it is believed that GA is an important regulator of young leaf development. However, the downstream mechanism involved in GA-dependent leaf development remains unknown.

We showed that PLA1 and PLA2 expression was positively regulated by GA. Previous studies indicated that both PLA1 and PLA2 expression is restricted in the leaf primordia, but not in the SAM (Miyoshi et al. 2004; Kawakatsu et al. 2006). This is consistent with GA synthesis in leaf primordia but not in the SAM. GA-related genes (GA3ox2, GA20ox2, SLR1) are expressed in young leaf primordial, including P0 and P0 primordia (Kaneko et al. 2003). This expression domain overlaps with that of PLA1 and PLA2, supporting the present finding that GA is involved in the regulation of PLA gene expression. The proposed primary function of PLA1 and PLA2 is precocious leaf maturation (Kawakatsu et al. 2006). Leaf maturation is a complex process involving organized expansion and differentiation of cells/tissues. Because GA also plays a role in this organized expansion and differentiation, it is thought that GA action in leaf primordia could be closely related to PLA1 and PLA2 functions. It is assumed that GA regulates leaf development through controlling PLA gene expression. Our in situ hybridization experiments revealed that expression of PLA1 was not only quantitatively enhanced, but was also ectopically expanded to the SAM in GA-signaling mutants and GA-applied plants. These indicate that GA also spatially regulates PLA1 and PLA2 expression.

In contrast, expression of GA biosynthetic, and GA-catabolizing and signaling genes was not significantly altered in pla1 and pla2 mutants. In addition, feedback regulation of GA-biosynthetic and GA-catabolizing genes after GA application was normal in pla1 and pla2 mutants. The rice DELLA protein, SLR1, is a key component of GA signaling, and is a principal factor responsible for feedback regulation of GA biosynthesis (Itoh et al. 2008; Yamaguchi 2008). The normal SLR1 expression level in the pla mutants suggests that SLR1-dependent GA signal transduction is operating normally.

Many GA-related and GA responsive genes have been identified; for example, PHYTOCHROME INTERACTING FACTOR in skotomorphogenesis, (Feng et al. 2008; de Lucas et al. 2008) and α-amylase genes in seed germination (Kaneko et al. 2002). In addition, several microarray experiments revealed many GA responsive genes (Yazaki et al. 2003; Yang et al. 2004; Jan and Komatsu 2006). In terms of leaf development, however, less is known about the downstream pathway of GA. Our analyses indicate that PLA1 and PLA2 are factors downstream of GA in leaves, and one action of GA is the PLA-dependent suppression of precocious leaf maturation.

Although the pla1 and pla2 leaf phenotypes were similar, PLA1 and PLA2 regulate leaf maturation process through independent genetic pathways. Indeed, a pla1 and pla2 double mutant showed a more severe phenotype than that of the single mutants (Kawakatsu et al. 2006). With regard to their molecular function, PLA1 encodes a member of the cytochrome P450 family thought to be involved in the biosynthetic pathway of an unknown substance (Miyoshi et al. 2004), and PLA2 encodes a MEI2-like RNA-binding protein that may interact with unidentified RNA molecules (Kawakatsu et al. 2006). Considering the independent expression regulation and molecular functions of PLA1 and PLA2, it would not be surprising if PLA1 and PLA2 have functionally diversified. The present results, however, show that both genes are regulated by GA and act downstream of GA signaling. The only difference between pla1 and pla2 was in the feedback regulation of GA-related genes. In pla1, the effect of GA3 and uniconazole treatment on GA20ox2 and GA2ox4 expression was almost identical to those in the wild type. In contrast, pla2 showed a weaker response to these treatments. This indicates that PLA2 is partially involved in the GA feedback mechanism, but PLA1 is not.