Description

Plant organ size control is an essential process of plant growth and development. The regulation of plant organ size involves a complicated network of genetic, molecular interactions, as well the interplay of environmental factors (Hepworth and Lenhlard, 2014). Two elements of plant organ size development are cell proliferation, resulting in increase in cell numbers, and cell expansion, which increases the overall organ size via larger cell area occupancy. However, our understanding of the fine-tuned regulation of organ size remains incomprehensive. Here, we report a temperature-sensitive hypocotyl elongation EMS-generated mutant, hereby referred to as elongated hypocotyl under high-temperature (elh). The elongated hypocotyl phenotype is prominent when the elh seedlings are grown at high temperature, 28^°C, but not under the growth temperature of 21^°C (Figure 1A). As hypocotyl elongation is driven primarily by cell elongation processes (Derbyshire et al. 2007), we compared the difference of organs size between in wild type and elh plants. We observed significantly larger organ sizes in elh plants, including cotyledons, petals and seeds when compared to the control (Student’s t-test p < 0.05, Figure 1B). We further inspected the cell size and number of these three organs in wild type and elh plants. In elh, the cell sizes in cotyledons and petals were significantly larger than wild type (Student’s t-test p < 0.05, Figure 1C). We also measured the cell density and organ area of cotyledons, petals and mature dissected embryos. By extrapolating cell numbers based on organ size, we found no differences in total cell numbers in either organ (Student’s t-test p > 0.05) indicating that cell expansion rather than cell proliferation was perturbed in elh. Plastochron, leaf initiation rate, has been previously linked to cell expansion (Wang et al. 2008). We observed reduced leaf number in elh plants when compared to wild type (Student’s t-test p < 0.05, Figure 1D) and a comprehensive analysis showed that elh plants produced leaves at a slower rate than wild type plants (Student’s t-test p < 0.05, Figure 1H), suggesting that perturbing the balance between cell division and cell expansion is linked to the developmental rate at which leaves are produced.

As auxin is a key hormone in regulating hypocotyl elongation and cell expansion (Gray et al. 1998; Chapman et al. 2012) and high auxin levels were shown to increase hypocotyl elongation (Wang et al. 2008; Zhao et al. 2001), we hypothesised that higher auxin levels in elh seedlings may cause the temperature-sensitive hypocotyl elongation phenotype. To test this hypothesis, we introduced the auxin inhibitor 1‐N‐naphthylphthalamic acid (NPA) into both wild-type and elh seedlings (Figure 1E). Higher levels of NPA reduced the hypocotyl length in both wild type and elh seedlings when grown at 28^°C. At the concentration of 1 µM NPA, the length of elh hypocotyls were similar to wild type with no NPA (Figure 1 E). When we applied exogeneous auxin, indole-3-acetic acid (IAA) to wild type and elh seedlings (Figure 1F), the hypocotyl length elongation response at 28^°C was similar in both genotypes (Figure 1F). Together these results suggest that elh accumulates higher auxin levels leading to a longer hypocotyl when compared to the wild type when grown at 28^°C.

To understand the underlying molecular cause of the observed elh phenotypes, we performed bulk segregant analysis combined with Next Generation Mapping (NGM) to identify the causative mutation. Our NGM analysis suggested that the causative mutation in elh resulted in a non-synonymous amino acid substitution from cysteine to tyrosine in the Ubiquitin carboxyl-terminal hydrolase 14 (UBP14; AT3G20630) gene (Figure 1G). UBP14 genetically complemented both the cotyledon size and plastochron phenotypes in transgenic elh plants (Student’s t-test p < 0.05, Figure 1H) demonstrating that UBP14 and ELH are the same gene. UBP14 is essential for early embryo development in Arabidopsis thaliana as null ubp14 mutants are embryo lethal (Doelling et al. 2001). Another UBP14 allele has been shown to regulate organ size via regulating cell cycle kinases which affect both cell numbers and cell expansion (Xu et al. 2016). The UBP14 allele, referred to as da3, has altered mRNA splicing resulting in a premature stop codon and the encoded protein lacks the conserved C-terminal His Box. Here, we characterized a novel UBP14 allele gene that regulates cell organ size and elongation of hypocotyl under high temperature in Arabidopsis. Based on our findings, we postulate that UBP14 regulates organ size via cell expansion through increased levels of auxin that affects the stability of HEAT SHOCK PROTEIN 90 (HSP90) dependent TIF1 co-receptor complexes. It has been shown that high temperatures lead to upregulation of auxin synthesis, followed by the activation of HSP90 to stabilize the TIR1 Auxin/IAA repressor degradation complex (Wang et al. 2016). The elh allele may act to destabilise the HSP90-TIR1 complex, leading to longer hypocotyls (Yamada et al.2009; Wang et al. 2016). Unlike the da3 mutant allele that perturbs both cell expansion and proliferation (Xu et al. 2016), our findings suggest that these two functions can be separated by altering protein function. The elh allele identified here is potentially a useful tool to understand the role of UBP14 in plant development.