visually in wheat by editing the sequence specific
toTacol-B5in Yangmai18 (notTaCol-B5in
CItr 17600, which is not yet transformable).
Damage to theTacol-B5 CCT domain in two
independent editing events,Tacol-B5-ED1
andTacol-B5-ED12 (Fig. 4, A and B), delayed
heading and reduced plant height (Fig. 4, C to
F). The edited CCT domain inTacol-B5 showed
effects on plant productivity in the greenhouse
but not in the field (fig. S15 and table S2),
suggesting thatTaCOL-B5 might regulate
multiple agronomic traits through its dif-
ferent domains ( 10 ).
We found that 10 sequenced wheat genomes
and five tetraploidT. durumwheat accessions
haveTacol-B5, whereas the tetraploid wild em-
mer wheat (T. turgidum) cultivar Zavitan has
TaCol-B5 (fig. S16). We developed a diagnos-
tic marker for the SNP involving the Ser^269 /
Gly^269 substitution betweenTaCol-B5 and
Tacol-B5 (fig. S17).TaCol-B5was found in
only 33 of 1657 accessions in a global collection
of modern wheat cultivars and germplasm
(table S4). It remains plausible that the rare
alleleTaCol-B5, accessible from modern wheat
cultivars in different continents, could be used
to enhance grain yield in a diverse array of
genetic backgrounds and environments.
Numerous CCT proteins have been found in
different plant species, and they are classified
into three families according to their domains:
COL, PRR (Pseudo-response regulator), and
CMF (CCT motif family) (Fig. 4G) ( 11 , 12 ). The
CCT proteins mostly regulate flowering through
pathways of photoperiod ( 13 – 15 ), circadian
rhythms ( 16 ), or vernalization ( 17 ), but a few
proteins, including ZmCCT in maize ( 18 ) and
Hd1 in rice ( 19 ),arereportedtobeinvolvedin
spike development.Ghd7in rice is a gene that
affects heading date, grain number, and plant
height, and Ghd7 protein does not have any
B-box or PRR (Fig. 4G) ( 20 ).TaCol-B5is not
orthologous toGhd7in sequence, and the
predicted full length of theTaCOL-B5 protein
has a conserved CCT domain (fig. S18) and
B1/B2-boxes (Fig. 4H and fig. S19, A and B). We
attempted to transform the same host plant
withTaCol-B5with the region encoding the
predicted B1/B2-boxes and without this region
to identify the functions of the B-boxes (fig. S19C),
but only the latter construct was successful
in transformation. The results demonstrated
that the expressedTaCol-B5 protein without
the predicted B-boxes was able to maintain
its functions in transgenic wheat. However,
the dominantTaCol-B5allele without the re-
gion encoding B-boxes was driven by the maize
ubiquitin promoter, which could produce pleio-
tropic effects in wheat ( 21 , 22 ). Future studies
should investigate functions of the predicted
B-boxes inTaCol-B5, oligomeric states and
structure ofTaCol-B5 proteins with or without
the B-boxes, and their downstream genes in
wheat plants. The clonedTaCol-B5has a gen-
eral role in promoting cell proliferation and
differentiation, leading to an overall increasein spikelet number and spike length, as well
as tiller and spike number and plant size, and
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ACKNOWLEDGMENTS
Funding:This project was supported by Agriculture and Food
Research Initiative Competitive Grants 2017-67007-25939,
2017-67007-25932, and 2022-68013-36439 from the USDA
National Institute of Food and Agriculture (NIFA). This project
was also supported by grants from the Oklahoma Center for
Advanced Science and Technology (OCAST, AR17-020-03), the
Oklahoma Wheat Research Foundation, the Oklahoma Agricultural
Experiment Station, and the Dillon and Lois Hodges Professorship.
H.J. received grants from the“ 111 ”Project and the Collaborative
Innovation Center for Modern Crop Production (CIC-MCP)
cosponsored by Province and Ministry, China. We thank E. Akhunov
for providing the pBUN421 plasmid and M. Tadege for valuable
discussion.Author contributions:X.Z. and H.J. performed
experimental procedures and analyzed results. T.L. contributed to
protein interaction and phosphorylation analyses. R.N., L.L., and
C.-C. K. contributed to gene expression, protein identification, and
greenhouse experiments. H.J., J.W., W.H., Z.L., C.P., and B.F.C.
contributed to field trials and phenotypic analyses. C.P. and C.C.
contributed to initial GBS marker development and QTL mapping.
L.Y. conceived of the idea, designed experiments, analyzed
genotypic and phenotypic data, and interpreted results. X.Z.,
H.J., and L.Y. wrote the manuscript. B.F.C. edited the manuscript.
Competing interests:The authors declare no competing interests.
Data and materials availability:All data are available in the
main text or the supplementary materials.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abm0717
Materials and Methods
Figs. S1 to S19
Tables S1 to S5
References ( 23 Ð 49 )
MDAR Reproducibility Checklist
Data S18 September 2021; accepted 22 February 2022
10.1126/science.abm0717SCIENCEscience.org 8 APRIL 2022•VOL 376 ISSUE 6589 183
Fig. 4. Functional domains and pleiotropic effects ofTaCOL-B5 proteins.(AtoF) Effects of edited
Tacol-B5. Edited sequences (in red) include a 1-bp deletion (Tacol-B5-ED1) (A) and a 7-bp deletion (Tacol-B5-ED12)
(B). Images were taken to show effects ofTacol-B5-ED1 on heading date (C) and plant height (D) and effects
ofTacol-B5-ED12 on heading date (E) and plant height (F). (G) Diagram of the structures of three plant CCT proteins.
M, methionine as the first amino acid at the N terminus. (H) Structure ofTaCol-B5 protein with the predicted
B1/B2-box.TaCol-B5-OE without the predicted B-boxes was tested in transgenic plants.
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