The HK5 and HK6 cytokinin receptors mediate diverse developmental pathways in rice

Abstract

The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice (Oryza sativa) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.

Keywords: Cell signaling; Cytokinin; Plant development; Plant hormones.

Fig. 1.

Effect of disruption of HK5 and HK6 on cytokinin responses. (A) Morphology of rice seedlings grown in the presence and absence of cytokinin. Wild-type or the indicated hk mutants were grown on Kimura B nutrient solution (Ma et al., 2001) solidified with 1% (w/v) gellan gum for 7 days in the presence of a vehicle control (0), 10 nM BA or 50 nM BA (6-benzylaminopurine) and representative seedlings photographed. (B-D) Quantification of primary root length (B), the number of lateral roots (C) and shoot length (D) in seedlings grown as in A. Letters indicate differences at a P<0.05 significance level using ANOVA analysis with a Tukey post-hoc correction. n≥15. (E,F) Fluorescence decline ratio (RFd) of wild-type (WT), hk5, hk6 and hk5 hk6 leaf sections in a dark-induced senescence assay. Dissected rice leaves were incubated for 3 days in the dark. RFd was measured before incubation (day 0) and 1 and 3 days after dark incubation. (E) Pseudo-coloring of leaves based on calculated RFd parameter at day 3 of the dark incubation. (F) RFd at day 0, 1 and 3 in the leaf sections from the indicated lines. Letters indicate differences at a P<0.05 significance level using ANOVA analysis with a Tukey post-hoc correction; lower-case blue letters (−BA samples) and red letters (+BA samples). ** indicates a significant difference (P<0.05) when samples without BA were compared to cognate +BA samples using a t-test. n≥9. (G) Quantification of type-A RR expression in response to cytokinin. Roots from 7-day-old wild-type or hk mutant seedlings were treated with 5 µM BA or a vehicle control for 1 h. Expression of RR2, RR4 and RR6 was quantified using qRT-PCR. The expression values were normalized to an ACT1 control gene and expressed as a fold-change relative to the vehicle control. Data represent the mean±s.e.m. of three biological replicates (n=3), each with three technical replicates.

Fig. 2.

Disruption of HKs alters root growth and development. (A) Adult root phenotypes of 9-week-old wild type and the indicated hk mutants grown in soil. The plants were removed from their pots, the soil gently washed off the roots and the plants photographed; representative images are shown. (B) Representative images of root apical meristems (RAMs) of seminal roots. Confocal microscopy images of fixed root tips from 14-day-old seedlings of the indicated genotypes grown in liquid Kimura B nutrient solution. The yellow arrows indicate the upper extent of the RAM in each root tip. (C) Quantification of RAM size for roots grown as in B. The RAM was measured from the quiescent center to the point where central xylem cells first elongate (yellow arrows in B). Values represent the mean±s.e.m. (n≥5). Individual data points are shown as gray circles. Letters indicate differences at a P<0.05 significance level using ANOVA analysis with a Tukey post-hoc correction. (D) Representative images of root tips of wild-type and the indicated hk mutants grown on Kimura B nutrient solution solidified with 1% (w/v) gellan gum. Roots were fixed and visualized via confocal microscopy. Scale bar: 100 µM. (E) Cross-sections of wild-type and hk mutant roots. Seedlings were grown for 5 days and the roots then embedded in agarose and stained with calcofluor white. Asterisks mark presumptive metaxylem cells. Scale bars: 50 µM.

Fig. 3.

Shoot and panicle phenotypes of hk mutants. (A) Representative images of adult plants. Wild-type and hk mutants were grown in soil for 12 weeks and representative plants photographed. The inset on the right is a close-up of hk5 hk6 tillers lacking panicles. Scale bar: 10 cm. (B) Representative images of mature panicles with flag leaf from 20-week-old plants showing panicle exsertion. (C-F) Quantification of leaf width (C), tiller number (D), panicle numbers (E) and the length of panicle exsertion (F) in 20-week old wild-type and the indicated hk mutant lines. Values represent the mean±s.e.m. n≥15 (C); n≥13 (D,E); n≥207 (F). (G) Representative images of isolated immature panicles from wild type and the indicated hk mutants. (H) Quantification of various aspects of panicle morphology in wild type and the indicated hk mutants (n≥51). For C-F and H, individual data points are shown as gray circles. Letters indicate differences at a P<0.05 significance level using an ANOVA analysis with a Tukey post-hoc correction.

Fig. 4.

The hk5 hk6 mutants exhibit altered panicle meristematic activity. (A) Longitudinal sections of wild-type (WT) and hk5 hk6 inflorescences collected at stage 4 of rice inflorescence development (Furutani et al., 2006). White triangles point to spikelet meristems (SMs). PA, primary axis (rachis); PB, primary branch; SB, secondary branch. Scale bars: 200 µm. (B) Expression of genes associated with panicle meristem development in 7-week-old wild-type and hk mutant plants, including both 7-week-old (hk5 hk6a) and 18-week-old (hk5 hk6b) hk5 hk6 plants. Letters indicate differences in gene expression at a P<0.05 significance level using ANOVA analysis with a Tukey post-hoc correction. Data represent the mean±s.e.m. of three biological replicates (n=3), each with two technical replicates.

Fig. 5.

Effects of hk mutations on floral and seed development. (A) Representative images of wild-type (WT) and the indicated hk mutant spikelets, all of which have had the palea removed in order to reveal inner whorl organs. Scale bar: 1 mm. (B) Quantification of the number of stamens per spikelet in wild-type and hk mutant spikelets (n≥50). (C) Quantification of the percentage of spikelets that were filled with grain in wild-type and hk mutants (n≥50). (D) Representative images of ten seeds from wild-type and the single hk mutants. (E) Quantification of the mean area of wild-type and hk mutant seeds (n≥20). (F) Representative images of wild-type and single hk mutant stigmas illustrating reduced brushes. (G) Quantification of the length of the brushes from wild-type and single hk mutant stigmas. n≥40. For B,C,E,G. letters indicate differences at a P<0.05 significance level using an ANOVA analysis with a Tukey post-hoc correction.