Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis.

KEY MESSAGE: We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides. We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52-54 kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

Translational control of phloem development by RNA G-quadruplex-JULGI determines plant sink strength.

The emergence of a plant vascular system was a prerequisite for the colonization of land; however, it is unclear how the photosynthate transporting system was established during plant evolution. Here, we identify a novel translational regulatory module for phloem development involving the zinc-finger protein JULGI (JUL) and its targets, the 5' untranslated regions (UTRs) of the SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs, which is exclusively conserved in vascular plants. JUL directly binds and induces an RNA G-quadruplex in the 5' UTR of SMXL4/5, which are key promoters of phloem differentiation. We show that RNA G-quadruplex formation suppresses SMXL4/5 translation and restricts phloem differentiation. In turn, JUL deficiency promotes phloem formation and strikingly increases sink strength per seed. We propose that the translational regulation by the JUL/5' UTR G-quadruplex module is a major determinant of phloem establishment, thereby determining carbon allocation to sink tissues, and that this mechanism was a key invention during the emergence of vascular plants.

Capsicum annuum homeobox 1 (CaHB1) is a nuclear factor that has roles in plant development, salt tolerance, and pathogen defense.

Homeodomain-leucine zipper (HD-Zip) family proteins are unique to plants, but little is known about their role in defense responses. CaHB1 is a nuclear factor in peppers, belonging to subfamily II of HD-Zip proteins. Here, we determined the role of CaHB1 in the defense response. CaHB1 expression was induced when pepper plants were challenged with Phytophthora capsici, a plant pathogen to which peppers are susceptible, or environmental stresses such as drought and salt stimuli. CaHB1 was also highly expressed in pepper leaves following application of SA, whereas ethephon and MeJA had a moderate effect. To further investigate the function of CaHB1 in plants, we performed gain-of-function study by overexpression of CaHB1 in tomato. CaHB1-transgenic tomatoes showed significant growth enhancement including increased leaf thickness and enlarged cell size (1.8-fold larger than control plants). Microscopic analysis revealed that leaves from CaHB1-transgenic plants had thicker cell walls and cuticle layers than those from controls. Moreover, CaHB1-transgenic plants displayed enhanced resistance against Phytophthora infestans and increased tolerance to salt stress. Additionally, RT-PCR analysis of CaHB1-transgenic tomatoes revealed constitutive up-regulation of multiple genes involved in plant defense and osmotic stress. Therefore, our findings suggest roles for CaHB1 in development, salt stress, and pathogen defense.

A novel pepper (Capsicum annuum) receptor-like kinase functions as a negative regulator of plant cell death via accumulation of superoxide anions.

Plant receptor-like kinases belong to a large gene family. The Capsicum annuum receptor-like kinase 1 (CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. The CaRLK1 extracellular domain (ECD)-green fluorescent protein (GFP) fusion protein was targeted to the plasma membrane, and the kinase domain of the CaRLK1 protein exhibited autophosphorylation activity. CaRLK1 transcripts were more strongly induced in treatment with Xag8ra than in treatment with Xag8-13. Furthermore, infection with incompatible Xanthomonas campestris pv. vesicatoria race 3 induced expression of CaRLK1 more strongly than in the compatible interaction. Cell death caused by both a disease-forming and an HR-inducing pathogen was delayed in the CaRLK1-transgenic plants. Ectopic expression of CaRLK1 also induced transcripts of the lesion stimulating disease (LSD) gene, a negative regulator of cell death. Respiratory burst oxidase homolog (RBOH) genes were up-regulated in the transgenic plants compared with the wild type, as the concentration of the superoxide anion was increased. In contrast, the concentration of H(2)O(2) did not differ between the transgenic and wild-type plants. These results support the theory that the suppression of plant cell death by CaRLK1 is associated with consistent production of the superoxide anion and induction of the RBOH genes and the LSD gene, but not with the concentration of H(2)O(2). Thus, CaRLK1 may be a receptor of an as yet unidentified pathogen molecular pattern and may function as a negative regulator of plant cell death.

NbHB1, Nicotiana benthamiana homeobox 1, is a jasmonic acid-dependent positive regulator of pathogen-induced plant cell death.

Induction of cell death is an important component of plant defense against pathogens. There have been many reports on the role of phytohormones in pathogen-induced cell death, but jasmonic acid (JA) has not been implicated as a regulator of the response. Here, we report the function of NbHB1, Nicotiana benthamiana homeobox1, in pathogen-induced cell death in connection with JA signaling. Involvement of NbHB1 in cell death was analysed by gain- and loss-of-function studies using Agrobacterium-mediated transient overexpression and virus-induced gene silencing, respectively. Expression of NbHB1 following pathogen inoculations and various treatments was monitored by reverse transcription polymerase chain reaction. Transcript levels of NbHB1 were upregulated by infection with virulent and avirulent bacterial pathogens. Ectopic expression of NbHB1 accelerated cell death following treatment with darkness, methyl jasmonate, or pathogen inoculation. Conversely, when NbHB1 was silenced, pathogen-induced cell death was delayed. NbHB1-induced cell death was also delayed by silencing of NbCOI1, indicating a requirement for JA-mediated signaling. Overexpression of the domain-deleted proteins of NbHB1 revealed that the homeodomain, leucine zipper, and part of the variable N-terminal region were necessary for NbHB1 functionality. These results strongly suggest the role of NbHB1 in pathogen-induced plant cell death via the JA-mediated signaling pathway.

S-nitrosylation: an emerging redox-based post-translational modification in plants.

S-nitrosylation, the covalent attachment of a nitric oxide moiety to a cysteine thiol, is now established as a key post-translational modification in animals. This process has been shown to regulate the function of a wide variety of regulatory, structural, and metabolic proteins. The emerging evidence now suggests that S-nitrosylation may also have a central function in plant biology.