Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses

Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed.Environmental stresses, including drought, high salinity, oxidative stress, and unfavorable temperatures, profoundly affect the growth and development of higher plants. Because of their sessile life cycle, plants have developed self-protective mechanisms to increase their tolerance to short- and long-term stresses by triggering diverse sets of signal transduction pathways and activating stress-responsive genes. The genetic and cellular mechanisms in response to abiotic stress have been widely documented in higher plants (Shinozaki and Yamaguchi-Shinozaki, 1996; Bray, 1997; Ishitani et al., 1997; Zhu, 2002; Bohnert et al., 2006; Shinozaki and Yamaguchi-Shinozaki, 2007; Vij and Tyagi, 2007).Lipid droplets (LDs) are dynamic subcellular organelles enclosed by a monolayer of phospholipid. LDs act as repositories for fatty acids and sterols, which are used for energy production and membrane and hormone synthesis. LDs are also involved in various cellular processes, including intracellular protein storage, stress responses, and lipid signaling (Bartz et al., 2007; Zehmer et al., 2009; Carman, 2012; Herker and Ott, 2012; Murphy, 2012; Sun et al., 2013; Kory et al., 2015). LDs bud from the endoplasmic reticulum (ER), where they become enriched with triacylglycerols and subsequently enlarged, until they pinch off to form an LD (Chapman et al., 2012; Chapman and Ohlrogge, 2012; Jacquier et al., 2013). Several reports suggest that LD-associated proteins, such as fat-specific protein 27 (FSP27), SEIPIN, and PERILIPIN1 (Plin1), are key regulators of LD formation in mammals, Drosophila, and yeasts (Farese and Walther, 2009; Xu et al., 2012; Yang et al., 2012). After budding from the ER, LDs fuse with each other and expand. In adipocytes, Plin1 functions as an enhancer of FSP27-mediated lipid transfer and LD growth, indicating that Plin1 and FSP27 participate in LD formation and fusion (Sun et al., 2013). Enlarged LDs provide surfaces to allow the attachment of numerous LD-associated proteins, which are later displaced during shrinkage of LDs by lipolysis (Kory et al., 2015).Because LDs are mainly present in seeds, studies on LD-associated proteins in higher plants have focused on seed development and germination (Chapman et al., 2013; Gidda et al., 2013; Horn et al., 2013; Szymanski et al., 2014). For example, oleosins regulate LD size in Arabidopsis (Arabidopsis thaliana) seed development (Siloto et al., 2006). Arabidopsis SEIPINs modulate LD proliferation and neutral lipid accumulation in developing seeds (Cai et al., 2015). On the other hand, the cellular roles of LD-associated proteins in postgermination growth remain largely unraveled.CaSRP1 (Capsicum annuum stress-related protein 1) was previously identified as a hot pepper small rubber particle protein (SRPPs) homolog (Hong and Kim, 2005). CaSRP1 was induced in response to water stress in hot pepper plants. Constitutive over-expression of CaSRP1 in transgenic Arabidopsis plants resulted in elevated growth and increased drought tolerance relative to wild-type Arabidopsis (Kim et al., 2010). CaSRP1 is evolutionarily related to SRPPs in rubber-producing plants (Wititsuwannakul et al., 2008). Rubber particles are single-membrane organelles that store rubber (cis-1,4-polyisoprene). Although rubber particles and LDs have different lipid compositions, their basic architectures are similar (Cornish et al., 1999). Thus, SRPP homologs may have common properties in the formation and biogenesis of rubber particles and/or LDs in rubber-producing and non-rubber-producing plants.In this report, we identified and characterized three SRPP homologs, SRP1, SRP2, and SRP3, in Arabidopsis. The SRP genes were differentially expressed in various tissues and induced by abscisic acid (ABA) and a broad spectrum of abiotic stress, including drought, high salinity, and low temperature. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis plants. In addition, ectopic expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines showed the opposite phenotypes. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. These results are discussed in light of the suggestion that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and drought stress response. The possible relationships between LD-associated proteins and stress tolerance response are also discussed.