MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.     

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets.     

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.     

Pipecolic Acid,an Endogenous Mediator of Defense Amplification and Priming,Is a Critical Regulator of Inducible Plant Immunity

Metabolic signals orchestrate plant defenses against microbial pathogen invasion. Here, we report the identification of the non-protein amino acid pipecolic acid (Pip), a common Lys catabolite in plants and animals, as a critical regulator of inducible plant immunity. Following pathogen recognition, Pip accumulates in inoculated Arabidopsis thaliana leaves, in leaves distal from the site of inoculation, and, most specifically, in petiole exudates from inoculated leaves. Defects of mutants in AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) in systemic acquired resistance (SAR) and in basal, specific, and β-aminobutyric acid–induced resistance to bacterial infection are associated with a lack of Pip production. Exogenous Pip complements these resistance defects and increases pathogen resistance of wild-type plants. We conclude that Pip accumulation is critical for SAR and local resistance to bacterial pathogens. Our data indicate that biologically induced SAR conditions plants to more effectively synthesize the phytoalexin camalexin, Pip, and salicylic acid and primes plants for early defense gene expression. Biological priming is absent in the pipecolate-deficient ald1 mutants. Exogenous pipecolate induces SAR-related defense priming and partly restores priming responses in ald1. We conclude that Pip orchestrates defense amplification, positive regulation of salicylic acid biosynthesis, and priming to guarantee effective local resistance induction and the establishment of SAR.