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Regulation of monocot and dicot plant development with constitutively active alleles of phytochrome B.

The constitutively active missense allele of Arabidopsis phytochrome B, AtPHYBY276H or AtYHB, encodes a polypeptide that adopts a light-insensitive, physiologically active conformation capable of sustaining photomorphogenesis in darkness. Here, we show that the orthologous OsYHB allele of rice phytochrome B (OsPHYBY283H ) also encodes a dominant "constitutively active" photoreceptor through comparative phenotypic analyses of AtYHB and OsYHB transgenic lines of four eudicot species, Arabidopsis thaliana, Nicotiana tabacum (tobacco), Nicotiana sylvestris and Solanum lycopersicum cv. MicroTom (tomato), and of two monocot species, Oryza sativa ssp. japonica and Brachypodium distachyon. Reciprocal transformation experiments show that the gain-of-function constitutive photomorphogenic (cop) phenotypes by YHB expression are stronger in host plants within the same class than across classes. Our studies also reveal additional YHB-dependent traits in adult plants, which include extreme shade tolerance, both early and late flowering behaviors, delayed leaf senescence, reduced tillering, and even viviparous seed germination. However, the strength of these gain-of-function phenotypes depends on the specific combination of YHB allele and species/cultivar transformed. Flowering and tillering of OsYHB- and OsPHYB-expressing lines of rice Nipponbare and Kitaake cultivars were compared, also revealing differences in YHB/PHYB allele versus genotype interaction on the phenotypic behavior of the two rice cultivars. In view of recent evidence that the regulatory activity of AtYHB is not only light insensitive but also temperature insensitive, selective YHB expression is expected to yield improved agronomic performance of both dicot and monocot crop plant species not possible with wild-type PHYB alleles.


Fig. 4G-H Are there images for OsYHB in Rc50?
Response: We don't have those images. In view of the whole plant phenotypes, OsYHB in Rc50 should have normal chloroplast development similar to that for wild-type rice in Rc50. For this reason, we did not secure these images. In view of the time to obtain these images, we have elected not to delay publication of this work at this time.

through RT-qPCR with appropriate controls, or by western blot (if the lab has access to a fluorescence imaging system and the antibodies for monocot and dicot YHB are compatible with dual labeling on a single blot).
Response: Unfortunately, we do not have an antibody that can quantitatively estimate the relative amounts of both monocot phyB and dicot phyB. For this reason, it is difficult to directly compare AtYHB and OsYHB protein level in the same species. Similarly, the DNA sequences of AtYHB and OsYHB differ so RT-qPCR estimates of transcript levels of both genes would also not be comparable. On the other hand, variation in YHB expression levels has very limited impact on the phenotype of transgenic plants, because phyB regulatory effects are nearly saturated once the transgene expression level is ~ 3 fold higher than the endogenous phyB level, according to an early study (Fig. 2 in Wagner et al 1996 Plant Cell 8:859-871) and our empirical experience in analyzing numerous transgenic plants overexpressing phyB variant alleles. We had dozens of independent 35S::OsYHB/Col lines that showed consistent longer hypocotyl than 35S::AtYHB/Col in darkness (a few lines are shown in Fig. 8B), so we conclude that the phenotypic difference is not due to systematic expression difference. For rice, we secured two homozygous 35S::AtYHB/Kit lines with highly consistent phenotypes, and in Fig. 8C immunoblot assay, one such line was confirmed to be strongly overexpressed. In summary, we do not think the weaker potency of cross-class overexpressed phyB compared to in-class one is due to variation in overexpression level. As such, we have not performed additional experiments to address this concern, but we have added text in the Discussion that addresses this possibility.
2) In the materials and methods, the explanation of qPCR conditions are insufficient to allow for reproduction. The authors should, as much as possible, follow MIQE guidelines (1. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., and Wittwer, C. T. "The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments." Clinical chemistry 55, no. 4 (2009): 611-622. doi:10.1373/clinchem.2008). The PCR conditions should be reported (e.g. Cycling conditions used) and if primer efficiency was considered when measuring differences in expression or perfect doubling was assumed. Response: The method section for quantitative RT-PCR has been substantially expanded to include full technical details.
Minor criticisms: 1.) In figures where coleoptiles, 1st and 2nd leaf sheaths are denoted, magenta is very close to red, and is difficult to distinguish. Could the authors choose another hue, or a different color to increase clarity of the figure. Response: Given that some readers may be red/green-blind, and to increase clarity of figures printed in dark/white mode, we have accordingly changed the red arrowheads in Figure 3, 8 and S4 to white to denote coleoptiles.

2.) Figure 6 A and B Natural is spelled incorrectly.
Response: So corrected. Figure 7B, which lines are measured? It would be nice to be able to compare with expression levels to the immunoblot in Fig 7A. Response: In the figure legend for 7B, lines #2 and #4 used for coleoptile measurement are now explicitly indicated. Figure 6C-F, the authors should run statistical analysis (ANOVA with multiple comparisons) on the measurements to illustrate at what point to the transgenic lines diverge from controls. Response: Thank you for this suggestion. We have accordingly performed statistical analysis on the data presented in Figure 6, not just panels C-F