Spatial and temporal expression of SlUVI4 andSlCCS52 genes in tomato hypocotyls
To understand the function of APC/C in tomato growth and development, we measured transcript abundance of SlUVI4 and SlCCS52s using quantitative reverse transcription–polymerase chain reaction (qRT-PCR).SlUVI4 had the highest expression level in stem and young fruit, and moderate expression in fully expanded leaf and ovary (Figure 3A).SlCCS52A1 had high expression levels in most organs except root and stamen, and SlCCS52A2 had relatively low expression levels in root, petal, sepal and young fruit (Figure 3B, C). SlCCS52B had similar tissue-specific expression patterns to SlUVI4 (Figure 3D). These data suggested that SlCCS52 genes are functionally redundant in the regulation of tomato organ growth and development. We then analyzed the transcription of SlUVI4 and SlCCS52genes in hypocotyls of day 1, 3, 5 and 7 after seed germination. All genes were highly expressed at day 1 followed by dramatically reduced transcripts afterwards (Figure 3E). The transcription ofSlCCS52A1 and SlCCS52A2 did not change at day 3 and 5, and had slight reduction and increase at day 7 (Figure 3E). The transcription of SlUVI4 and SlCCS52B had significant reductions at day 5 and 7 compared to day 3. Meanwhile, we measured the transcript abundances of APC/C core genes, SlAPC2 andSlAPC11 . Both SlAPC2 and SlAPC11 had dramatic reduction of transcript abundances at day 3, and restored the transcription afterwards (Figure S4). Taken together, these data indicate that APC/C activity is differentially regulated bySlUVI4 and SlCCS52 genes in various tissues, andSlUVI4 and SlCCS52B may have more specific roles in the regulation of hypocotyl elongation.
Lights repress hypocotyl elongation throughSlUVI4 andSlCCS52B mediated cell cycle progression.
To investigate light effects on tomato hypocotyl growth, we measured hypocotyl length and diameter, and cell cycle progression in hypocotyl cells of cultivar ‘Heinz 1706’ under dark and different light conditions. The hypocotyl length was significantly longer under dark condition than white light condition, while the hypocotyl diameter was significantly thicker under white light condition (Figure S5A-C, E-G). Cell cycle progression in hypocotyl cells was consistent in the dark, resulting in relatively steady endoreduplication indices (EIs) (Figure S5D). Under white light condition, cell cycle progression in hypocotyl cells varied at different days, in which the proportion of 2C nuclei increased gradually after day 1 resulting in reduction of EI values (Figure S5H). Overall, EI values in the white light were smaller than that in the dark, which correlated with hypocotyl elongation. We then measured transcript abundances of SlUVI4 and SlCCS52 , and all of them had highest transcription at day 1 under both dark and light conditions (Figure S6). The transcription of SlUVI4 andSlCCS52B gradually reduced during the hypocotyl elongation under light condition, which correlated with cell cycle progression. We further investigated effects of light qualities on hypocotyl growth, cell cycle progression and transcription of SlUVI4 andSlCCS52 genes. The hypocotyls under red light were significantly longer than that under blue light whereas diameters of hypocotyls gradually increased under both light conditions (Figure 4A-C, E-G). The proportion of 2C nuclei gradually increased under both light conditions resulting in reduction of EI values, which was more pronounced under red light condition (Figure 4D, H). qRT-PCR analyses revealed that the transcription of SlUVI4 and SlCCS52B gradually reduced under both light conditions, which was parallel to changes in cell cycle progression (Figure 4I). Their transcript abundance accumulated more under red light than blue light condition, which correlated with longer hypocotyls under red light condition. We observed the reduction ofSlCCA52A1 not SlCCS52A2 transcription during the hypocotyl growth, and SlCCS52A1 expression was slightly higher under red light, suggesting weak roles of SlCCS52A1 in hypocotyl elongation under different light conditions. All these data above suggest thatSlUVI4 and SlCCS52B play important roles in hypocotyl elongation through the regulation of cell cycle progression.
MS salts and sugar enhance hypocotyl elongation throughSlUVI4 and SlCCS52B mediated cell cycle progression.
To further understand roles of SlUVI4 and SlCCS52B in tomato hypocotyl elongation, we checked whether their expressions were correlated with hypocotyl elongation under Murashige and Skoog (MS) salts and sugar treatments. Without MS salts in the growth medium (non-MS, NMS), the average hypocotyl lengths were 8.5, 18.6, 21.9 and 25.4 mm, and the average diameters of hypocotyls were 0.62, 0.76, 0.69 and 0.80 mm at day 1, 3, 5 and 7 (Figure 5A, B, C). With MS salts in the growth medium (MS), the average hypocotyl lengths were 9.5, 31.3, 35.9 and 39.2 mm, and the average diameters of hypocotyls were 0.76, 0.92, 0.81 and 0.89 mm at day 1, 3, 5 and 7 (Figure 5E, F, G). With MS salts and sugar both in the growth medium (MSS), the average hypocotyl lengths were 5.3, 28.1, 37.7 and 49.2 cm, and the average diameters of hypocotyls were 0.72, 0.89, 0.87 and 1.06 mm at day 1, 3, 5 and 7 (Figure 5I, J, K). These data above indicated that addition of MS salts with or without sugar significantly promoted hypocotyl elongation and thickening. Cell cycle analyses revealed that 16C nuclei only appeared in seedlings grown on MS and MS with sugar growth medium (Figure 5H, L), which resulted in the increase of EI values compared to that on non-MS medium (Figure 5D). In fact, addition of MS salts with sugar significantly increased abundances of 8C and 16C nuclei leading to higher EI values. qRT-PCR analyses of transcription of SlUVI4 and SlCCS52 genes in hypocotyls at day 7 showed that MS with or without sugar had highest and moderate transcriptions ofSlUVI4 and SlCCS52B , and only MS with sugar could enhance the transcription of SlCCS52A1 and SlCCS52A2 (Figure 5M). All these data suggested that MS salts with or without sugar promoted hypocotyl elongation possibly through SlUVI4 and SlCCS52Bcoordinated cell cycle progression.