Os03g0790600

The rice gene Os03g0790600 was reported as PLASTOCHRON3 (PLA3)/GOLIATH (GO) in 2009.

Annotated Information

Function

• PLA3/GO encodes a glutamate carboxypeptidase II and is an ortholog of Arabidopsis AMP1 and maize VP8.

• PLA3 regulates cell proliferation in various tissues.

• PLA genes regulate leaf maturation and SAM maintenance.

• PLA genes are involved in plant hormone homeostasis.

Mutation

• Two recessive mutations, pla3-1 and pla3-2, caused pleiotropic phenotypes including a shortened plastochron (Figures 1a). Mature embryos of pla3/go mutants were significantly larger than those of wild type (Figure 1b–e). All of the embryonic organs were enlarged, including scutellum, plumule, radicle and epiblast (Figure 1d,e). The patterning and positioning of the organs were not disturbed. In pla3-3 (go-1) and pla3-4 (go-2), two radicles were occasionally formed (Figure 1h). In addition, four foliage leaves were differentiated in pla3 embryos in contrast to three leaves in wild-type embryos (Figure 1f,g). The overproduction of leaves could be due to a reduced dormancy, because pla3 seeds frequently showed vivipary (Figure 1i–k).

Figure 1. Seedlings and embryos of wild type and pla mutants. (a) Seedlings of wild type, pla1-4, pla2-1 and pla3-1 at 3 weeks after germination. (b, d, f, i) Wild type. (c, e, g, h, j, k) pla3. (b, d) Mature seeds. The embryo of pla3 is markedly larger than that of wild type. (d–g) Longitudinal sections of mature embryos. (f, g) Close-up views of the area within the red square in (d and e), respectively. In the wild type, three foliage leaves are formed, in contrast, four foliage leaves are present in pla3. Parts are numbered in order of initiation. (i, j) Top views of a dormant embryo of wild type (i) and a viviparous one of pla3 (j). (k) Viviparous seed of pla3. Bar = 5 cm in (a), 2.5 mm in (b and c), 200 lm in (d–g), 500 lm in (h), 1 mm in (i and j) and 2 mm in (k). .

• In the vegetative phase, abnormalities were mainly observed in shoots. Although roots were morphologically normal, gravitropism was slightly reduced. The researchers examined cell divisions in pla3 plants surviving more than a month by in situ hybridization using histone H4 specifically expressed in the S-phase of the cell cycle. In median longitudinal sections of pla3 SAM, many more histone H4 signals were detected than in the wild type (5.2 ± 2.0 in pla3-1, n = 9 versus 0.7 ± 0.3 in wild type, n = 10) (Figure 2a,b). This value was larger than those in pla1 and pla2 (1.9 ± 0.7 in pla1-4, n = 10 and 3.8 ± 1.7 in pla2-1, n = 10) (Kawakatsu et al., 2006). Since the cell size of pla3-1 plants was not significantly altered (23.7 ± 6.2 lm in pla3-1 mature leaves versus 20.5 ± 4.7 in wild-type mature leaves), accelerated cell divisions are thought to directly contribute to the shortened plastochron in pla3. Simultaneous measurement of the shape and size of the SAM revealed that the pla3 SAM is larger than the wild-type SAM (Table 1).

Figure 2. Vegetative phenotypes of wild type and pla3. (a, e, l, n) Wild type. (b–d), (f), (g–k), (m), (o–q) pla3-1. (a, b) Expression of histone H4 in the shoot apex. (c) Lethal pla3-1seedling at 2 weeks after germination. (d) Longitudinal section of the shoot apex in (c). A filamentous leaf primordium (arrow) is present in place of the shoot apical meristem (SAM). (e, f) Expression of OSH1 in the shoot apex. OSH1 is down-regulated in the P0 region. (g, h) Elongated SAM (arrowheads). (i) Closely initiated leaves fused into the X-shaped leaf and sharing one midvein (arrow). (j) Cleared image of SAM division. Two SAMs are present (arrowheads). (k) Scanning electron microscopy image of a divided SAM. One SAM grows robustly (arrow), but the other grows weakly (arrowhead). (l, m) Cross-section of shoot apex. Asterisks indicate differentiating procambial strands. Overlaps of two leaf margins (arrowheads) are observed at the P2 stage in wild type and the P3 stage in pla3. (n, o) Longitudinal sections of the shoot apex. A ligule primordium protrusion is formed at the P3 leaf primordium (arrows). (p) Leaf with jagged margin. (q) Scanning electron microscopy image of jagged leaf margin. The leaf margin is not torn physically. Bars = 50 lm in (a, b, e, f), 500 lm in (c), 100 lm in (d, l–o), 120 lm in (g), 5 mm in (h), 250 lm in (i, q), 200lm in (j), 1 mm in (k) and 2 mm in (p). .

Table 1 Phenotypes of wild type, and pla mutants in vegetative phase (±SD) .

• In rice, the transition between the vegetative and reproductive phases is accompanied by substantial elongation of the upper four or five internodes. pla3 showed severe dwarfism (<10% of wild-type plant height), but the number of elongated internodes was increased to eight except for the uppermost one (Figure 3a,b). Non-elongation of the uppermost internode and the increase in the number of elongated internodes were commonly observed in pla1, pla2 and pla3 (Figure 3a,b). At the heading stage, pla3 mutants produced ectopic shoots instead of panicle, as did pla1 and pla2 (Figure 3c). The number of ectopic shoots in pla3-1 (4.3; n = 10) was larger than in pla1-4 and pla2-1 (3.2, 2.5, respectively; n = 10). In pla3, after the formation of the flag leaf, primary branch primordia were formed normally in 2/5 spiral phyllotaxy, but they developed as vegetative shoots (Figure 3d,e). This conversion was always associated with over-growth of the bracts that were normally aborted without elongation (Figure 3c). These reproductive phenotypes commonly observed in pla1, pla2 and pla3 suggest that vegetative and reproductive programs are co-expressed in pla mutants.

Figure 3. Reproductive phenotypes of wild type, pla1, pla2 and pla3. .

Expression

• To further understand the function of PLA3 we examined its expression pattern. First, we performed semi-quantitative RT-PCR to reveal the organ-specific expression pattern. Total RNA was isolated from embryos at 10 days after pollination (DAP), from vegetative shoot apices at 3 weeks after germination, and from leaf blades, roots, inflorescence apices and spikelets. PLA3 was expressed uniformly in all organs examined (Figure 5), in contrast to PLA1 and PLA2 that are expressed specifically in young leaf primordia and the inflorescence apex .

• Next, the researchers analyzed the detailed expression pattern of PLA3 by in situ hybridization with digoxigenin (DIG)-labeled antisense RNA PLA3 probe. PLA3 transcripts were detected throughout the whole plant body except the endosperm, suggesting that PLA3 was expressed ubiquitously. In a control experiment we could not detect any expression with sense RNA PLA3 probe. The researchers detected strong PLA3 expression in the epidermal layer of the scutellum of 10-DAP embryos and in spikelet meristems.

Evolution

• Rice, maize and Arabidopsis GPCIIs were classified into two subfamilies. One comprises PLA3, VP8 and AMP1, and the other consists of the several other GPCIIs (Figure 4).

Figure 4. Phylogenetic tree of glutamate carboxypeptidases. Numbers at each branch point indicate bootstrap values (n = 1000). .

Labs working on this gene

• Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan,
• DuPont Crop Genetics Research, Experimental Station, PO Box 80353, Wilmington, DE 19880-0353, USA, and
• RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan