TILLERS ABSENT1, the WUSCHEL ortholog, is not involved in stem cell maintenance in the shoot apical meristem in rice

ABSTRACT

Stem cell maintenance in the shoot apical meristem (SAM) is very important for plant development and is regulated by the WUSCHEL–CLAVATA (WUS–CLV) feedback loop in Arabidopsis (Arabidopsis thaliana). WUS promotes stem cell identity, whereas CLV negatively regulates stem cell proliferation by repressing WUS expression. We previously showed that, in rice (Oryza sativa), the WUS ortholog TILLERS ABSENT1 (TAB1, also known as OsWUS) has no function in SAM maintenance, whereas it plays a crucial role in axillary meristem development. Recently, we showed that a double mutant of FLORAL ORGAN NUMBER2 (FON2) and ABERRANT SPIKELET AND PANICLE1 (ASP1) led to a marked enlargement of the inflorescence meristem, and that the TAB1 function is not associated with massive stem cells in this meristem. In this paper, we confirmed that TAB1 is also unrelated to the enlargement of the SAM in the vegetative phase of the fon2 and fon2 asp1 mutants. In addition, misexpression of TAB1 under the promoter of FON1 led to a slight reduction of the SAM size in wild type, suggesting that TAB1 is not a positive regulator of stem cells. Taking together, TAB1 seems not to be involved in meristem maintenance, irrespective of the meristem type.

The shoot apical meristem (SAM), which is a reservoir of stem cells, plays crucial roles in plant development. Stem cell homeostasis is maintained by the WUSCHEL–CLAVATA (WUS–CLV) feedback loop in Arabidopsis (Arabidopsis thaliana).^1,2 WUS promotes stem cell fate, whereas CLV signaling negatively regulates stem cell proliferation. In rice (Oryza sativa), a pathway similar to CLV signaling is reported to regulate the maintenance of the flower meristem.^2,3 The FLORAL ORGAN NUMBER1 and FON2 genes encode CLV1-like LRR-receptor kinase and CLV3-like CLE protein, respectively.^4,5 Mutations in either FON1 or FON2 lead to an enlargement of the flower meristem due to overproliferation of stem cells, resulting in increases of floral organs, such as carpels and stamens. Recently, our close observation indicated that the fon2 mutation also causes an enlargement of the SAM in the vegetative phase.⁶ In addition, we revealed that ABERRANT SPIKELET AND PANICLE1 (ASP1) encoding a TOPLESS (TPL)-like corepressor is involved in stem cell homeostasis by negatively regulating a set of genes in concert with FON signaling.⁶ The inflorescence meristem is markedly enlarged in the fon2 asp1 double mutant, showing massive overproliferation of stem cells. By contrast, loss of function of the WUS ortholog, TILLERS ABSENT1 (TAB1, also known as OsWUS), does not affect the maintenance of the SAM; instead, axillary shoot formation was strongly compromised in the tab1 mutant, suggesting that TAB1 plays a critical role in axillary meristem development.⁷ In this paper, we reexamined the role of TAB1 in meristem maintenance in the vegetative phase, using fon2 and asp1 mutants and transgenic plants misexpressing TAB1 in the SAM.

The shoot apex of the rice seedling was cleared by treating with a solution (chloral hydrate:water:glycerol = 8:3:1) for at least 48 hours. We observed the SAM of wild type, fon2, asp1, tab1, and their double and triple mutants (Figure 1(a–h)), and measured the heights of the SAMs to quantitatively compare the sizes of the SAMs of these strains (Figure 1(i)). Neither tab1 nor asp1 showed any difference in SAM size, compared to th wild type (Figure 1(a,c,d,i)). The result showing no statistically significant difference in the SAM size between tab1 and wild type confirmed our morphological observation that the tab1 mutation does not affect SAM size.⁷ The SAM size of fon2 was slightly but significantly larger than that of wild type, whereas the fon2 asp1 double mutant produced a larger SAM than did the fon2 single mutant (Figure 1((a,b,e,i)), confirming our previous result that the asp1 mutation enhances the fon2 phenotype.⁶ If the enlargement of the SAM is related to TAB1 activity in fon2 and fon2 asp1, the SAM size should be reduced by the genetic introduction of the tab1 mutation into these mutants. However, the result indicated that no significant changes were observed in meristem size between fon2 and fon2 tab1, and between fon2 asp1 and fon2 asp1 tab1 (Figure 1). Therefore, it is unlikely that the TAB1 function is associated with SAM enlargement in the fon2 and fon2 asp1 mutants. Probably, TAB1 has no role in the maintenance of the SAM. These results are greatly in contrast with the fact that the wus mutation causes premature termination of the SAM and is epistatic to the clv3 mutation in Arabidopsis.^8,9

Figure 1.

Effects of the tab1 mutation on the SAM size.

(a–h) Shoot apices of wild type (a), fon2-3 (b), asp1-10 (c), tab1-1 (d), fon2-3 asp1-fe (e), fon2-3 tab1-1 (f), asp1-10 tab1-1 (g), and fon2-3 asp1-fe tab1-1 (h). Scale bars = 50 µm. (i) Quantification of the height of the SAM. n = 15 (wild type), 13 (fon2-3), 15 (asp1-10), 15 (tab1-1), 15 (fon2-3 asp1-fe), 15 (fon2-3 tab1-1), 3 (asp1-10 tab1-1), 15 (fon2-3 asp1-fe tab1-1). Different letters (a, b, c, and d) indicate significant differences between samples (P < 0.05, Tukey’s test).

We next examined the effect of the misexpression of TAB1 under the promoter of FON1, which is expressed in the SAM.⁵ We observed the SAM about three months after regeneration. The pFON1-TAB1 plants showed a dome-shaped SAM, similar to the control plants. However, the SAM size was seemingly reduced in the pFON1-TAB1 plants, as compared with the control plants (Figure 2(a,b). Although the sample size was small, the measurement of SAM height indicated that the SAM size was statistically significantly reduced in the pFON1-TAB1 plants (p < 0.05), as compared with the control plants (Figure 2(f)). The plant size, which was mainly affected by the size and the number of leaves, was also decreased in the pFON1-TAB1 plants, although the extent of the decrease varied among the plants (Figure 2(c–e)). These abnormalities may be associated with the smaller SAM in the pFON1-TAB1 plants. Thus, it seems likely that misexpression of TAB1 in the SAM does not have a positive effect on the meristem.

Figure 2.

Effects of pFON1-TAB1.

(a, b) The SAM in the cleared shoot apex. Scale bars = 50 µm. (c–e) Plants, three months after regeneration. Transgenic plants with pFON1-GUS (a, c) and pFON1-TAB1 (b, d, e). (f) The size of the SAMs. Transgenic plants with pFON1-GUS (gray) and pFON1-TAB1 (blue). Welch’s t-test; p = 0.035 (height), p = 0.088 (width). n = 9 (pFON1-GUS), 3 (pFON1-TAB1).

In this paper, we showed that the tab1 mutation did not bring about any effects on the SAM size, not only in the wild type, but also in the fon2 and fon2 asp1 mutants, in which stem cells were overproliferated. These results agree with our previous conclusion that TAB1 has no function in the SAM.⁷ Taking together with our recent finding that the tab1 mutation does not affect enlarged inflorescence meristems of fon2 and fon2 asp1 mutants,⁶ TAB1 seems to have no function in stem cell maintenance in rice, irrespective of the type of meristem. Unexpectedly, misexpression of TAB1 in the SAM appeared to cause a slight reduction of the meristem size. In rice, OsWOX4, a paralog of TAB1, reportedly promotes stem cell identity.¹⁰ One possible explanation for the size reduction of the SAM in pFON1-TAB1 plants is that misexpression of TAB1 would inhibit the OsWOX4 function indispensable for stem cell promotion, probably due to competition in binding to target genes and/or interacting with protein partners. Therefore, we concluded that TAB1, unlike Arabidopsis WUS, is not likely to play a key role in SAM maintenance in rice.

Funding Statement

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) under Grant 23248001; the Japan Society for the Promotion of Science (JSPS) under Grant 16J04197. C.S. was supported by JSPS for a Research Fellowship for Young Scientists.