Os10g0403000

Rice PLASTOCHRON genes

Annotated Information

Function

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. Concomitantly, leaves of pla1 become short, suggesting that PLA1 regulates organ size. The PLA1 gene encodes a cytochrome P450 family protein (CYP78A11). An Arabidopsis homolog of PLA1, KLUH, was shown to regulate organ size. Subsequently, the PLA2 and PLA3 genes, loss-of-function mutants of which exhibit similar phenotypes to that of pla1, were identified. 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. 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 primordial through shoot meristems to suppress the formation of a new leaf primordium. Thus, PLA1 and PLA2 are key genes for elucidating leaf development.

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. These mutant phenotypes suggest that PLA genes have some relationship with phytohormones.

Fig. 1. Phenotypes of wild-type, plal mutant, and transgenicplal-2 plants. (a) Seedlings of wild-type (WT) and pla -2 plants 17 days after germination, showing that many more leaves are formed in plal-2 than in wild type. Arrowheads indicate lamina joint. (b) Panicles of wild type, plal-1 with vegetative shoots instead of primary branches, and plal-2 with truncated panicle, one shoot (arrowhead), and enlarged bract (arrow). (c) Scanning electron microscopy of a wild-type young panicle. (d) Scanning electron microscopy of a plal-1 young panicle. Asterisks indicate ectopic vegetative shoots with an enlarged bract. (e) plal-2-like transgenic plants carrying pBGH1 alone. Normal transgenic plants carrying pBGH1/P450 are shown to the right. (f) Panicles of transgenic plants: left, a plal-2-like panicle with enlarged bract (arrowhead) in transgenic plant carrying pBGH1; right, normal panicle in transgenic plant carrying pBGH1/P450.

Expression

PLA1 and PLA2 gene expression is associated with GA signaling. Thus, the research team examined the effect of GA on PLA1 and PLA2 gene expression. Ten-day-old seedlings were treated with 10 lM 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. To investigate the longterm effect of GA, wild-type seeds were inoculated and grown for 8 days on culture media containing 10 lM GA3. PLA1 and PLA2 expression was maintained at a high level for 8 days. Next, we examined the effect of uniconazole, a GA biosynthesis inhibitor. Uniconazole treatment markedly suppressed PLA1 and PLA2 gene expression. Therefore, GA regulates the expressions of both PLA1 and PLA2.

Fig. 2. In situ expression of PLA1 in vegetative and reproductive apex of wild-type plant. Dark blue stains represent PLA gene expression. (a) Median longitudinal section of shoot apex 1 month after germination. (b) Schematic representationo f a. (c) Longitudinals ection of shoot apex just after transition to reproductive phase. Two bracts of primary branches are formed. Arrows indicate PLA1 expression in the internodes of an elongating stem. (d) Longi-tudinal section of a young panicle at a slightly later stage of c. (e) Longitudinal section of a developing panicle in which spikelets are being formed. Arrows indicate PLA1 expression in the rachis internodes. (f) Longitudinal section of young spikelet. PO,p lastochronOle af founder cells; P1, plastochronl leaf; P2, plastochron2 leaf; P3, plastochron3 leaf; Br, bract; Ibr, incipient bract; Rm, rachis meristem; Pr, Primary branch primordium; Fm, floral meristem; Le, lemma primordium; Eg, empty glume primordium; Rg, rudimentary glume primordium.

Expression of GA-related genes in pla 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.

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. 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 ofGAslightly decreased the expression of GA20ox2 in wild-type, pla1-4 and pla2-1 plants (Fig. 3b). In contrast, uniconazole treatment markedly enhanced expression in wild type and pla1-4, and moderately enhanced it in pla2-1, plants (Fig. 3b). 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. 3c). In pla2-1 plants, GA also induced the expression, but to a limited extent (Fig. 3c). Uniconazole treatment suppressed GA2ox4 expression in wild-type, pla1-4 and pla2-1 plants (Fig. 3c).

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.

Fig. 3. 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

PLA gene expression in GA-related mutants

PLA1 and PLA2 expression in d18 was slightly down-regulated compared with the wild type (Fig. 4a). 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. 4b).

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. 4c–e).This ectopic expression of PLA1 coincided with that induced by GA treatment. 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.

Fig. 4. 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 wildtype shoot apex (c) and slr1-1 shoot apex (d, e). Arrows indicate ectopic expression. Bars 100 lm

Evolution

According to the recommendation of the Cyto-chrome P450 Gene Nomenclature Committee, the PLA1 protein was designated as CYP78A11 (GenBank accession no. AB096259). The CYP78A class comprises 11 members including rice CYP78A11. Rice CYP78A11 forms a cluster with maize CYP78A1 (GenBank accession no. L23209) and Arabidopsis CYP78A7 (GenBank accession no. AC016893). CYP78A11 exhibits the highest similarity to the CYP78A1 of maize (71% amino acid identity), and strong similarity to Arabidopsis CYP78A7 (61% identity) and Pinus radiata CYP78A4 (59% identity; GenBank accession no. AF049067). Genes in the CYP78A class belong to the group A cytochrome P450 in plants. They are phylogenetically more closely related to each other than to non-group A cytochrome P450s and seem to be involved in plant-specific reactions.

Fig. 5. Phylogenetic relationship among CYP78A proteins. The phylogenetic tree was generated based on the entire amino acid sequences by using the CLUSTAL Wprogram

Labs working on this gene

Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

Department of Biology, Aichi University of Education, Kariya 448, Japan

Plant Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan

References

1 Manaki Mimura;Yasuo Nagato;Jun-Ichi Itoh, Rice PLASTOCHRON genes regulate leaf maturation downstream of the gibberellin signal transduction pathway, Planta, 2012, 235(5): 1081-1089

2 Kazumaru Miyoshi;Byung-Ohg Ahn;Taiji Kawakatsu;Yukihiro Ito;Jun-Ichi Itoh;Yasuo Nagato;Nori Kurata, PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450, Proceedings of the National Academy of Sciences, 2004, 101(3): 875-880

3 Jun-Ichi Itoh;Atsushi Hasegawa;Hidemi Kitano;Yasuo Nagato, A Recessive Heterochronic Mutation, plastochron1, Shortens the Plastochron and Elongates the Vegetative Phase in Rice, The Plant Cell, 1998, 10(9): 1511-1522