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. 2016 Mar;26(3):384-8.
doi: 10.1038/cr.2015.116. Epub 2015 Sep 25.

NF-YB1-regulated expression of sucrose transporters in aleurone facilitates sugar loading to rice endosperm

Ai-Ning Bai  1   2 , Xiao-Duo Lu  1 , Dong-Qi Li  1   2 , Jin-Xin Liu  1 , Chun-Ming Liu  1
Affiliations

NF-YB1-regulated expression of sucrose transporters in aleurone facilitates sugar loading to rice endosperm

Ai-Ning Bai et al. Cell Res. 2016 Mar.
No abstract available

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Figures

Figure 1
Figure 1
NF-YB1-regulated expression of three sucrose transporters facilitates grain filling in rice. (A) Real-time PCR analysis showing that NF-YB1 is expressed in caryopses at 4, 7, 11, 14 and 21 DAP; and in caryopses collected at 11 DAP, NF-YB1 is highly expressed in mixed aleurone and testa tissues. l, leaves; r, roots; p, panicles; o, ovaries; c2 to c31, caryopses collected at 2 to 31 DAP; se and a+t, starchy endosperm and mixed aleurone and testa samples collected at 11 DAP, respectively. Data are shown as means ± SD (n = 3). (B) In situ hybridization in sectioned caryopses collected at 2, 4, 5, 9 and 11 DAP using antisense (upper panel) and sense probes (lower panel), showing that NF-YB1 is strongly expressed in aleurone (arrowheads), but not in maternal tissues, or in starchy endosperm. dv, dorsal vascular bundle; np, nucellar projection. Bar = 100 μm. (C) nfyb1-1 and nfyb1-2 knockout mutants generated by CRISPR-Cas9. Upper panel, a gene model of NF-YB1 to show positions of single-nucleotide insertions in nfyb1-1 and nfyb1-2. Lower panel, mutation sites in nfyb1-1 and nfyb1-2, as compared to WT sequences. Sequence in bold, target sites; underlined, protospacer-adjacent motif sequences (PAMs). Arrowheads indicate inserted single nucleotides. (D) Mature grains of nfyb1-1 and nfyb1-2, showing the chalky endosperm phenotype as compared to the semi-transparent endosperm in WT. Upper panel, side views of intact caryopses; lower panel, surface views of cracked caryopses. en, endosperm; em, embryo. Bars = 1 mm. (E) Scanning electron microscopy images of cracked mature WT and nfyb1-1 caryopses at different magnifications, showing smaller and loosely packed starch grains in nfyb1-1, as compared to large and tightly packed ones in the WT. Scale bars: 500 μm (top), 25 μm (middle), 5 μm (bottom). (F) Sucrose, fructose, and glucose contents (μg in mg fresh weight) in developing caryopses collected at 6, 9 and 11 DAP, showing significantly reduced accumulation of all these sugars in nfyb1-1, as compared to that in the WT caryopses at the same stage. Data shown as means ± SD (n = 3; *P< 0.05, **P< 0.01, based on Student's t-test). (G) Expression levels of SUT1, SUT3, SUT4 and MST4 were reduced in nfyb1-1 caryopses collected at 5 DAP, as compared to those in WT. Expression levels of these genes in the WT were normalized to 1. No significant reduction was observed for SUT2. Data shown as means ± SD (n = 3). (H) Yeast one-hybrid assay to show bindings of NF-YB1 to SUT1, SUT3, SUT4 and MST4 regulatory elements. NF-YB1 fused to GAL4 transcriptional activation domain (GAD-NF-YB1) was used as prey; 5′ regulatory sequences of SUT1, SUT3, SUT4, MST4 and SUT2 fused to LacZ as baits. Yeast cells co-transformed with GAD-NF-YB1 (left) or empty vector (GAD, right) and one of these baits were grown on a synthetic dropout medium without tryptophan and uracil. Note no activation was detected for SUT2. (I) LUC assays performed in rice protoplasts to show NF-YB1 activated SUT1, SUT3 and SUT4 promoters. LUC activities in protoplasts co-transfected with p35S::tNOS and reporter constructs (pSUT1::LUC, pSUT3::LUC, pSUT4::LUC, pMST4::LUC or pSUT2::LUC) were normalized to 1, and LUC activities in protoplasts co-transfected with p35S::NF-YB1::tNOS and these reporter constructs were relative to this value. Data shown as means ± SD (n = 3; *P< 0.05, **P< 0.01, based on Student's t-test). (J) EMSA assays to show His-tagged NF-YB1 bound directly to CCAAT box-containing 5′ upstream sequences of SUT1, SUT3 and SUT4. SUT4Δ, SUT4 with a mutated CCAAT box was used as a negative control. (K) A working model, illustrating the role of NF-YB1 in regulating sucrose loading to developing rice endosperms by activating three sucrose transporters in aleurone, while glucose and fructose are loaded to endosperms through an independently regulated route. DP, dorsal phloem; PC, parenchyma cells in the vascular bundle; NP, nucellar projection; DA, dorsal aleurone; SE, starchy endosperm; SP, symplastic; AP, apoplastic; suc, sucrose; glu, glucose; fru, fructose.
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