Uncovering DELLA-Independent Gibberellin Responses by Characterizing New TomatoproceraMutants

Abstract

Gibberellin (GA) regulates plant development primarily by triggering the degradation/deactivation of the DELLA proteins. However, it remains unclear whether all GA responses are regulated by DELLAs. Tomato (Solanum lycopersicum) has a single DELLA gene named PROCERA (PRO), and its recessive pro allele exhibits constitutive GA activity but retains responsiveness to external GA. In the loss-of-function mutant pro DGRAS , all examined GA developmental responses were considerably enhanced relative to pro and a defect in seed desiccation tolerance was uncovered. As pro, but not pro DGRAS , elongation was promoted by GA treatment, pro may retain residual DELLA activity. In agreement with homeostatic feedback regulation of the GA biosynthetic pathway, we found that GA20oxidase1 expression was suppressed in pro DGRAS and was not affected by exogenous GA 3 . In contrast, expression of GA2oxidase4 was not affected by the elevated GA signaling in pro DGRAS but was strongly induced by exogenous GA 3 . Since a similar response was found in Arabidopsis thaliana plants with impaired activity of all five DELLA genes, we suggest that homeostatic GA responses are regulated by both DELLA-dependent and -independent pathways. Transcriptome analysis of GA-treated pro DGRAS leaves suggests that 5% of all GA-regulated genes in tomato are DELLA independent. INTRODUCTIONThe phytohormone gibberellin (GA) regulates numerous developmental processes throughout the plant life cycle, including seed germination, stem elongation, flowering, and fruit set (Yamaguchi, 2008). The signaling pathway from GA perception to transcriptional activation has been intensively studied over the past two decades and its major components have been identified. The nuclear DELLA proteins, a subgroup of the GRAS transcription factors family, suppress GA signaling (Locascio et al., 2013). GA binding to the soluble GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor triggers GID1 interaction with the DELLA proteins (Ueguchi-Tanaka et al., 2005;Nakajima et al., 2006;Griffiths et al., 2006), which then stimulates assembly of the DELLA proteins into an SCF E3 ubiquitin ligase complex via the GID2/SLEEPY1 F-box proteins. The SCF complex polyubiquitinates the DELLA proteins, targeting them for destruction by the 26S proteosome (Sasaki et al., 2003;Dill et al., 2004;Griffiths et al., 2006;Harberd et al., 2009;Hauvermale et al., 2012). GA, via GID1, can also reduce DELLA activity through a degradation-independent mechanism (Ariizumi et al., , 2013Ueguchi-Tanaka et al., 2008).Despite the central role of DELLAs in GA signaling, the mechanism underlying this regulation is not fully understood. Several studies have shown that protein-protein interactions play a major role in DELLA function. DELLAs bind to various transcription factors and proteins affecting transcription, including PHYTOCHROME-INTERACTING FACTORs (PIFs), ALCATRAZ, MYC2, JASMONATE-ZIM-DOMAIN PROTEIN9, SCARECROW LIKE3 (SCL3), and TCP transcription factors (de Lucas et al., 2008;Feng et al., 2008;Arnaud et al., 2010;Ga...

Functional analysis of the seven in absentia ubiquitin ligase family in tomato

“…In addition, the petal tip of the abnormal 35S::SlSINA5 flower was curled up to greater level than that of the normal tomato flower; and the abnormal 35S::SlSINA5 flower was overall smaller in size: The length of sepal, petal, stamen, or style was reduced to certain degree (Figure b), with about 39% reduced in stamen and 16% reduced in style (Figure c). Thus, the exserted stigma of the abnormal 35S::SlSINA5 flower was attributable to reduced size of the stamen rather than to elongation of the style, which is a novel phenotype found in tomato mutants with altered floral morphology (Carrera, Ruiz‐Rivero, Peres, Atares, & Garcia‐Martinez, ; García‐Hurtado et al ., ; Livne et al ., ). Significantly, because of the protruded stigma, such defective flowers were not able to self‐pollinate naturally for fruit‐set.…”

Cytokinin activity increases stomatal density and transpiration rate in tomato

“…RNA extraction and cDNA synthesis were performed as described by Livne et al (2015). Quantitative reverse transcription-PCR analysis was performed using the Absolute Blue qPCR SYBR Green ROX Mix (AB-4162/B) kit (Thermo Fisher Scientific).…”

Using CRISPR/Cas9 genome editing in tomato to create a gibberellin‐responsive dominant dwarf DELLA allele

“…The previously reported PROCERA mutant phenotype was due to a T905A mutation leading to the conversion of VHVID to VHEID in the C-terminal GRAS motif, which is thought to be important for DELLA action but has no defined biochemical function. A PRO mutant allele was obtained using TALENs in order to determine the role of PROCERA in GA signalling (Livne et al, 2015). In contrast with the first characterized PROCERA allele which carries a missense mutation and shows partial responsiveness to GA, isolated proTALEN and proΔGRAS mutations are complete null alleles and GA insensitive (Livne et al, 2015).…”

“…GA binding to the soluble Gibberellin Insensitive Dwarf (GID) receptor triggers GID interaction with the DELLA proteins, which then stimulates the assembly of an SCF E3 ubiquitin ligase complex that contains the GID2/SLEEPY1 F-box proteins (Ueguchi-Tanaka et al, 2005). This SCF complex polyubiquitinates the DELLA proteins, leading to their degradation by the 26S proteasome, thus derepressing growth (Livne et al, 2015). The A. thaliana genome encodes five highly homologous DELLA proteins including GA-insensitive (GAI) and repressor of ga1-3 (RGA) (Peng et al, 1997).…”