Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.Plastids originated from the endosymbiosis of cyanobacteria and can differentiate into several forms depending on their intracellular functions during the plant life cycle (Sakamoto et al., 2008). The amyloplast is a terminally differentiated plastid responsible for starch synthesis and storage. Starch forms insoluble particles in amyloplasts, referred to as starch grains (SGs). SGs are easily visualized by staining with iodine solution, and they can be observed using a light microscope. SGs are observed in storage organs such as seed endosperm, potato (Solanum tuberosum) tubers, and pollen grains. Nonstorage tissues such as endodermis and root caps also contain SGs (Morita, 2010).Cereal endosperm accumulates high levels of starch in amyloplasts. The volume of SGs is approximately the same as the volume of amyloplasts that fill most of the intracellular space. SGs in rice (Oryza sativa) endosperm are normally 10 to 20 μm in diameter (Matsushima et al., 2010). One amyloplast contains a single SG that is assembled of several dozen smaller starch granules. Each starch granule is a sharp-edged polyhedron with a typical diameter of 3 to 8 μm. This type of SG is called a compound SG (Tateoka, 1962). For compound SGs, starch granules are assembled (but not fused) to form a single SG, which is easily separated by conventional purification procedures. By contrast, simple SGs contain a single starch granule. Simple SGs are produced in several important crops, such as maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and wheat (Triticum aestivum; Tateoka, 1962; Matsushima et al., 2010, 2013).The size of SGs in cereal endosperm is diverse. Maize and sorghum SGs have a uniform size distribution of approximately 10 μm in diameter (Jane et al., 1994; Matsushima et al., 2010; Ai et al., 2011). In barley and wheat, SGs of two discrete size classes (approximately 15−25 μm and less than 10 μm) coexist in the same cells (Evers, 1973; French, 1984; Jane et al., 1994; Matsushima et al., 2010). In Bromus species, intrageneric size variations of SGs are observed in which even phylogenetic neighbors develop distinctly sized SGs (Matsushima et al., 2013). The size of SGs can be controlled by manipulating the activity of starch synthetic enzymes using transgenic plants or genetic mutants (Gutiérrez et al., 2002; Bustos et al., 2004; Ji et al., 2004; Stahl et al., 2004; Matsushima et al., 2010). However, the molecular mechanism that controls the interspecific size variations of SGs has not been resolved.The SG occupies most of the amyloplast interior, because the SG is approximately the same size as the amyloplast. The size of amyloplasts may affect the size of SGs, or vice versa. Amyloplasts and chloroplasts both develop from proplastids. The size of chloroplasts is controlled by the chloroplast binary fission division machinery, especially by the ring structures that form at the division sites (Miyagishima, 2011). Proteins involved in the ring structures have been isolated, including Filamenting temperature-sensitive mutantZ (FtsZ), Minicell locusD (MinD), MinE, and ACCUMULATION AND REPLICATIONS OF CHLOROPLAST5 (ARC5). Arabidopsis (Arabidopsis thaliana) mutants that are defective in these proteins have defects in chloroplast division and contain enlarged and dumbbell-shaped chloroplasts. In contrast to the binary fission of chloroplasts, amyloplasts divide at multiple sites and generate a beads-on-a-string structure (Yun and Kawagoe, 2009). The inhibition of the chloroplast division machinery does not result in enlarged amyloplasts (Yun and Kawagoe, 2009).We recently developed a rapid method to prepare thin sections of endosperm (Matsushima et al., 2010). Using this method, SGs in mature endosperm can be easily and clearly observed. We performed genetic screening for rice mutants defective in SG morphology and size. One of the isolated mutants, substandard starch grain4 (ssg4), develops enlarged SGs in its endosperm. In this study, we characterized ssg4 phenotypes and identified the responsible gene. SSG4 encodes a protein containing 2,135 amino acid residues and an N-terminal plastid-targeted sequence. The domain of unknown function 490 (DUF490) was found at the C terminus of SSG4, where the ssg4 mutation was located. This suggests that SSG4 is a novel factor that influences the size of SGs and has potential as a molecular tool for starch breeding and biotechnology.     

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a d_N/d_S-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.Receptor-like kinases (RLKs) constitute one of the largest gene families in plants and expanded massively in land plants (Embryophyta; Lehti-Shiu et al., 2009, 2012). For plant RLK gene families, the functions of most members are often not known (especially in recently expanded families), but some described functions include innate immunity (Albert et al., 2010), pathogen response (Dodds and Rathjen, 2010), abiotic stress (Yang et al., 2010), development (De Smet et al., 2009), and sometimes multiple functions (Lehti-Shiu et al., 2012). The RLKs usually consist of three domains: an N-terminal extracellular domain, a transmembrane domain, and a C-terminal kinase domain (KD). In plants, the KD usually has a Ser/Thr specificity (Shiu and Bleecker, 2001), but Tyr-specific RLKs were also described (e.g. BRASSINOSTEROID INSENSITIVE1; Oh et al., 2009). Interestingly, it was estimated that approximately 20% of RLKs contain a catalytically inactive KD (e.g. STRUBBELIG and CORYNE; Chevalier et al., 2005; Castells and Casacuberta, 2007; Gish and Clark, 2011). In Arabidopsis (Arabidopsis thaliana), 44 RLK subgroups (SGs) were defined by inferring the phylogenetic relationships between the KDs (Shiu and Bleecker, 2001). Interestingly, different SGs show different duplication/retention rates (Lehti-Shiu et al., 2009). Specifically, RLKs involved in stress responses show a high number of tandemly duplicated genes whereas those involved in development do not (Shiu et al., 2004), which suggests that some RLK genes are important for the responses of land plants to a changing environment (Lehti-Shiu et al., 2012). There seem to be relatively few RLK pseudogenes compared with other large gene families, and copy retention was argued to be driven by both drift and selection (Zou et al., 2009; Lehti-Shiu et al., 2012). As most SGs are relatively old and RLK subfamilies expanded independently in several plant lineages, duplicate retention cannot be explained by drift alone, and natural selection is expected to be an important driving factor in RLK gene family retention (Lehti-Shiu et al., 2009).Leucine-rich repeat-receptor-like kinases (LRR-RLKs), which contain up to 30 leucine-rich repeat (LRRs) in their extracellular domain, constitute the largest RLK family (Shiu and Bleecker, 2001). Based on the KD, 15 LRR-RLK SGs have been established in Arabidopsis (Shiu et al., 2004; Lehti-Shiu et al., 2009). So far, two major functions have been attributed to them: defense against pathogens and development (Tang et al., 2010b). LRR-RLKs involved in defense are predominantly found in lineage-specific expanded (LSE) gene clusters, whereas LRR-RLKs involved in development are mostly found in nonexpanded groups (Tang et al., 2010b). It was also discovered that the LRR domains are significantly less conserved than the remaining domains of the LRR-RLK genes (Tang et al., 2010b). In addition, a study of four plant genomes (Arabidopsis, grape [Vitis vinifera], poplar [Populus trichocarpa], and rice [Oryza sativa]) showed that LRR-RLK genes from LSE gene clusters show significantly more indications of positive selection or relaxed constraint than LRR-RLKs from nonexpanded groups (Tang et al., 2010b).The genomes of flowering plants (angiosperms) have been shown to be highly dynamic compared with most other groups of land plants (Leitch and Leitch, 2012). This dynamic is mostly caused by the frequent multiplication of genetic material, followed by a complex pattern of differential losses (i.e. the fragmentation process) and chromosomal rearrangements (Langham et al., 2004; Leitch and Leitch, 2012). Most angiosperm genomes sequenced so far show evidence for at least one whole-genome multiplication event during their evolution (Jaillon et al., 2007; D’Hont et al., 2012; Tomato Genome Consortium, 2012). At a smaller scale, tandem and segmental duplications are also very common in angiosperms (Arabidopsis Genome Initiative, 2000; International Rice Genome Sequencing Project, 2005; Rizzon et al., 2006). Although the most common fate of duplicated genes is to be progressively lost, in some cases they can be retained in the genome, and adaptive as well as nonadaptive scenarios have been discussed to play a role in this preservation process (for review, see Moore and Purugganan, 2005; Hahn, 2009; Innan, 2009; Innan and Kondrashov, 2010). Whole-genome sequences also revealed that the same gene may undergo several rounds of duplication and retention. These LSE genes were shown to evolve under positive selection more frequently than single-copy genes in angiosperms (Fischer et al., 2014). That study analyzed general trends over whole genomes. Here, we ask if, and to what extent, this trend is observable at LRR-RLK genes. As this gene family is very dynamic and large, and in accordance with the results of Tang et al. (2010b), we expect the effect of positive selection to be even more pronounced than in the whole-genome average.We analyzed 33 Embryophyta genomes to investigate the evolutionary history of the LRR-RLK gene family in a phylogenetic framework. Twenty LRR-RLK SGs were identified, and from this data set, we deciphered the evolutionary dynamics of this family within angiosperms. The expansion/reduction rates were contrasted between SGs and species as well as in ancestral branches of the angiosperm phylogeny. We then focused on genes whose number increased dramatically in an SG- and/or species-specific manner (i.e. LSE genes). Those genes are likely to be involved in species-specific cellular processes or adaptive interactions and were used as a template to infer the potential occurrence of positive selection. This led to the identification of sites at which positive selection likely acted. We discuss our results in the light of angiosperm genome evolution and current knowledge of LRR-RLK functions. Positive selection footprints identified in LSE genes highlight the importance of combining evolutionary analysis and functional knowledge to guide further investigations.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.Polyamines (PAs) are low-molecular mass aliphatic amines that are present in almost all living organisms. Cellular PA concentrations are governed primarily by the balance between biosynthesis and catabolism. In plants, the major PAs are the diamine putrescine (Put), the triamine spermidine (Spd), and the tetraamines spermine (Spm) and thermospermine (T-Spm; Kusano et al., 2008; Alcázar et al., 2010; Mattoo et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014). Put is synthesized from Orn by Orn decarboxylase and/or from Arg by three sequential reactions catalyzed by Arg decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Arabidopsis (Arabidopsis thaliana) does not contain an ORNITHINE DECARBOXYLASE gene (Hanfrey et al., 2001) and synthesizes Put from Arg via the ADC pathway. Put is further converted to Spd via an aminopropyltransferase reaction catalyzed by spermidine synthase (SPDS). In this reaction, an aminopropyl residue is transferred to Put from decarboxylated S-adenosyl-Met, which is synthesized by S-adenosyl-Met decarboxylase (SAMDC; Kusano et al., 2008). Spd is then converted to Spm or T-Spm, reactions catalyzed in Arabidopsis by spermine synthase (SPMS; encoded by SPMS) or thermospermine synthase (encoded by Acaulis5 [ACL5]), respectively (Hanzawa et al., 2000; Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010). A recent review reports that T-Spm is ubiquitously present in the plant kingdom (Takano et al., 2012).The PA catabolic pathway has been extensively studied in mammals. Spm and Spd acetylation by Spd/Spm-N¹-acetyltransferase (Enzyme Commission no. 2.3.1.57) precedes the catabolism of PAs and is a rate-limiting step in the catabolic pathway (Wallace et al., 2003). A mammalian polyamine oxidase (PAO), which requires FAD as a cofactor, oxidizes N¹-acetyl Spm and N¹-acetyl Spd at the carbon on the exo-side of the N⁴-nitrogen to produce Spd and Put, respectively (Wang et al., 2001; Vujcic et al., 2003; Wu et al., 2003; Cona et al., 2006). Mammalian spermine oxidases (SMOs) perform oxidation of the carbon on the exo-side of the N⁴-nitrogen to produce Spd, 3-aminopropanal, and hydrogen peroxide (Vujcic et al., 2002; Cervelli et al., 2003; Wang et al., 2003). Thus, mammalian PAOs and SMOs are classified as back-conversion (BC)-type PAOs.In plants, Spm, T-Spm, and Spd are catabolized by PAO. Plant PAOs derived from maize (Zea mays) and barley (Hordeum vulgare) catalyze terminal catabolism (TC)-type reactions (Tavladoraki et al., 1998). TC-type PAOs oxidize the carbon at the endo-side of the N⁴-nitrogen of Spm and Spd to produce N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, plus 1,3-diaminopropane and hydrogen peroxide (Cona et al., 2006; Angelini et al., 2008, 2010). The Arabidopsis genome contains five PAO genes, designated as AtPAO1 to AtPAO5. Four recombinant AtPAOs, AtPAO1 to AtPAO4, have been homogenously purified and characterized (Tavladoraki et al., 2006; Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012). AtPAO1 to AtPAO4 possess activities that convert Spm (or T-Spm) to Spd, called partial BC, or they convert Spm (or T-Spm) first to Spd and subsequently to Put, called full BC. Ahou et al. (2014) report that recombinant AtPAO5 also catalyzes a BC-type reaction. Therefore, all Arabidopsis PAOs are BC-type enzymes (Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012; Ahou et al., 2014). Four of the seven PAOs in rice (Oryza sativa; OsPAO1, OsPAO3, OsPAO4, and OsPAO5) catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a), whereas OsPAO7 catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO2 and OsPAO6 remain to be characterized, but may catalyze TC-type reactions based on their structural similarity with OsPAO7. Therefore, plants possess both TC-type and BC-type PAOs.PAs are involved in plant growth and development. Recent molecular genetic analyses in Arabidopsis indicate that metabolic blocks at the ADC, SPDS, or SAMDC steps lead to embryo lethality (Imai et al., 2004; Urano et al., 2005; Ge et al., 2006). Potato (Solanum tuberosum) plants with suppressed SAMDC expression display abnormal phenotypes (Kumar et al., 1996). It was also reported that hydrogen peroxide derived from PA catabolism affects root development and xylem differentiation (Tisi et al., 2011). These studies indicate that flux through metabolic and catabolic PA pathways is required for growth and development. The Arabidopsis acl5 mutant, which lacks T-Spm synthase activity, displays excessive differentiation of xylem tissues and a dwarf phenotype, especially in stems (Hanzawa et al., 2000; Kakehi et al., 2008, 2010). An allelic ACL5 mutant (thickvein [tkv]) exhibits a similar phenotype as that of acl5 (Clay and Nelson, 2005). These results indicate that T-Spm plays an important role in Arabidopsis xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012).Here, we demonstrate that Arabidopsis pao5 mutants contain 2-fold higher T-Spm levels and exhibit aerial tissue growth retardation approximately 50 d after sowing compared with that of wild-type plants. Growth inhibition of pao5 stems and leaves at an early stage of development is induced by growth on media containing low T-Spm concentrations. Complementation of pao5 with AtPAO5 rescues T-Spm-induced growth inhibition. We confirm that recombinant AtPAO5 catalyzes BC of T-Spm (or Spm) to Spd. Our data strongly suggest that endogenous T-Spm levels in Arabidopsis are fine tuned, and that AtPAO5 regulates T-Spm homeostasis through a T-Spm oxidation pathway.     

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene

The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope ¹³C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO₂]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.Isoprenoids represent the largest and most diverse group (over 50,000) of natural compounds and are essential in all living organisms (Gershenzon and Dudareva, 2007; Thulasiram et al., 2007). They are economically important for humans as flavor and fragrance, cosmetics, drugs, polymers for rubber, and precursors for the chemical industry (Chang and Keasling, 2006). The broad variety of isoprenoid products is formed from two building blocks, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP). In plants, the plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Zeidler et al., 1997) produces physiologically and ecologically important volatile organic compounds (VOCs), the carotenoids (tetraterpenes; Giuliano et al., 2008; Cazzonelli and Pogson, 2010), diterpenes, the prenyl side-chains of chlorophylls (Chls) and plastoquinones, isoprenylated proteins, the phytohormones gibberellins, and side-chain of cytokinins (for review, see Dudareva et al., 2013; Moses et al., 2013). Industrially important prokaryotes (e.g. Escherichia coli) also use the MEP pathway for the biosynthesis of isoprenoids (Vranová et al., 2012), and there is an increasing interest in manipulating the MEP pathway of engineered microbes to increase production of economically relevant isoprenoids (Chang and Keasling, 2006). To achieve this, a mechanistic understanding of the regulation of the MEP pathway is needed (Vranová et al., 2012).Some plants, including poplars (Populus spp.), produce large amounts of the hemiterpene VOC isoprene. Worldwide isoprene emissions from plants are estimated to be 600 teragrams per year and to account for one-third of all hydrocarbons emitted to the atmosphere (Arneth et al., 2008; Guenther, 2013). Isoprene has strong effects on air chemistry and climate by participating in ozone formation reactions (Fuentes et al., 2000), by prolonging the lifespan of methane, a greenhouse gas (Poisson et al., 2000; Archibald et al., 2011), and by taking part in the formation of secondary organic aerosols (Kiendler-Scharr et al., 2012).Poplar leaves invest a significant amount of recently fixed carbon in isoprene biosynthesis (Delwiche and Sharkey, 1993; Schnitzler et al., 2010; Ghirardo et al., 2011) to cope with abiotic stresses (Sharkey, 1995; Velikova and Loreto, 2005; Behnke et al., 2007, 2010b, 2013; Vickers et al., 2009; Loreto and Schnitzler, 2010; Sun et al., 2013b), although there are indications that other protective mechanisms can partially compensate the lack of isoprene emission in genetically transformed poplars (Behnke et al., 2012; Way et al., 2013). It has been suggested that in isoprene-emitting (IE) species, most of the carbon that passes through the MEP pathway is used for isoprene biosynthesis (Sharkey and Yeh, 2001). However, a recent study using pulse-chase labeling with ¹⁴C has shown continuous synthesis and degradation of carotenes and Chl a in mature leaves of Arabidopsis (Arabidopsis thaliana; Beisel et al., 2010), and the amount of flux diverted to carotenoid and Chl synthesis compared with isoprene biosynthesis in poplar leaves is not known.Isoprene emission is temperature, light, and CO₂ dependent (Schnitzler et al., 2005; Rasulov et al., 2010; Way et al., 2011; Monson et al., 2012; Li and Sharkey, 2013a). It has been demonstrated that isoprene biosynthesis depends on the activities of IDP isomerase (EC 5.3.3.2), isoprene synthase (ISPS; EC 4.2.3.27), and the amount of ISPS substrate, DMADP (Brüggemann and Schnitzler, 2002a, 2002b; Schnitzler et al., 2005; Rasulov et al., 2009b). In turn, DMADP concentration has been hypothesized to act as a feedback regulator of the MEP pathway by inhibiting 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), the first enzyme of the MEP pathway (Banerjee et al., 2013). Understanding the controlling mechanism of isoprene biosynthesis is not only of fundamental relevance, but also necessary for engineering the MEP pathway in various organisms and for accurate simulation of isoprene emissions by plants in predicting atmospheric reactivity (Niinemets and Monson, 2013).There is ample evidence that silencing the ISPS in poplar has a broad effect on the leaf metabolome (Behnke et al., 2009, 2010a, 2013; Way et al., 2011; Kaling et al., 2014). While some of those changes (e.g. ascorbate and α-tocopherol) are compensatory mechanisms to cope with abiotic stresses, others (e.g. shikimate pathway and phenolic compounds) might be related to the alteration of the MEP pathway (Way et al., 2013; Kaling et al., 2014). The perturbation of these metabolic pathways can be attributed to the removal of a major carbon sink of the MEP pathway and the resulting change in the energy balance within the plant cell (Niinemets et al., 1999; Ghirardo et al., 2011). In this work, we analyzed the carbon fluxes through the MEP pathway into the main plastidic isoprenoid products.We used the ¹³C-labeling technique as a tool to measure the carbon fluxes through the MEP pathway at different temperatures, light intensities, and CO₂ concentrations in mature leaves of IE and transgenic, isoprene-nonemitting (NE) gray poplar (Populus × canescens). Isoprene emission was drastically reduced in the transgenic trees through knockdown of PcISPS gene expression by RNA interference, resulting in plants with only 1% to 5% of isoprene emission potential compared with wild-type plants (Behnke et al., 2007).We measured the appearance of ¹³C in the isoprenoid precursors 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (MEcDP) and DMADP as well as isoprene and the major downstream products of the MEP pathway, i.e. carotenoids and Chls. To reliably detect de novo synthesis of the pigments, which occur at very low rates (Beisel et al., 2010), we used isotope ratio mass spectrometry (IRMS).Here, (1) we quantify the effect of isoprene biosynthesis on the MEP pathway in poplar, and (2) we show that suppression of isoprene biosynthesis negatively affects the carbon flux through the MEP pathway by accumulating plastidic DMADP, which feeds back to inhibit PcDXS, leading to (3) a slight increase of carbon flux toward production of greater chain-length isoprenoids and (4) a strong decrease in the overall isoprenoid carbon fluxes to compensate for the much lower MEP pathway demand for carbon. This study strongly supports the hypothesis that an important regulatory mechanism of the MEP pathway is the feedback regulation of plastidic DMADP on DXS. The large carbon flux through the MEP pathway of IE poplar plastids demonstrates the potential of transgenically altered IE plant species to produce economically valuable isoprenoids at high rates in, for instance, industrial applications.     

A Chimeric Arabinogalactan Protein Promotes Somatic Embryogenesis in Cotton Cell Culture

Arabinogalactan proteins (AGPs) are a family of extracellular plant proteoglycans implicated in many aspects of plant growth and development, including in vitro somatic embryogenesis (SE). We found that specific AGPs were produced by cotton (Gossypium hirsutum) calli undergoing SE and that when these AGPs were isolated and incorporated into tissue culture medium, cotton SE was promoted. When the AGPs were partly or fully deglycosylated, SE-promoting activity was not diminished. Testing of AGPs separated by reverse-phase high-performance liquid chromatography revealed that the SE-promoting activity resided in a hydrophobic fraction. We cloned a full-length complementary DNA (cotton PHYTOCYANIN-LIKE ARABINOGALACTAN-PROTEIN1 [GhPLA1]) that encoded the protein backbone of an AGP in the active fraction. It has a chimeric structure comprising an amino-terminal signal sequence, a phytocyanin-like domain, an AGP-like domain, and a hydrophobic carboxyl-terminal domain. Recombinant production of GhPLA1 in tobacco (Nicotiana tabacum) cells enabled us to purify and analyze a single glycosylated AGP and to demonstrate that this chimeric AGP promotes cotton SE. Furthermore, the nonglycosylated phytocyanin-like domain from GhPLA1, which was bacterially produced, also promoted SE, indicating that the glycosylated AGP domain was unnecessary for in vitro activity.Arabinogalactan proteins (AGPs) comprise a diverse group of plant proteoglycans (for review, see Fincher et al., 1993; Nothnagel, 1997; Seifert and Roberts, 2007; Ellis et al., 2010). They are structurally complex, generally consisting of a Pro-, Ala-, Ser-, and Thr-rich protein backbone that is extensively modified, principally by hydroxylation of Pro residues (to Hyp) and subsequent glycosylation through O-linkages with type II arabinogalactans (Tan et al., 2003; Shimizu et al., 2005). Many AGPs also have a C-terminal hydrophobic domain that is processed and replaced with a glycosylphosphatidylinositol (GPI) anchor, which acts to tether the molecule to the extracellular face of the plasma membrane (Schultz et al., 1998). AGPs are also defined by their ability to be bound and precipitated by the synthetic dye β-glucosyl Yariv reagent (β-GlcY) and related molecules (Yariv et al., 1967). These dyes have been useful in isolating, localizing, and quantifying AGPs.AGPs are grouped into three subclasses (Schultz et al., 2002): AGPs have an N-terminal signal sequence, an arabinogalactosylated domain, and a hydrophobic C-terminal domain; “chimeric AGPs” contain at least one arabinogalactosylated domain and a domain with an unrelated motif; while “hybrid AGPs” contain arabinogalactosylated as well as different Pro/Hyp-rich glycoprotein motifs.AGPs are implicated in many aspects of plant cell growth and development. Historically, it was not possible to assign roles to individual AGPs, as tests were conducted with unfractionated mixtures of AGPs. More recently, individual AGPs, mainly from Arabidopsis (Arabidopsis thaliana), have been studied using techniques such as mutant analysis and gene knockout/silencing, providing evidence for roles of individual AGPs in cell expansion, root and seed regeneration, the coordination of vascular development, both male and female gametogenesis, the development of cotton fibers, and as contributors to plant stem strength (Shi et al., 2003; van Hengel and Roberts, 2003; Acosta-García and Vielle-Calzada, 2004; Motose et al., 2004; Yang et al., 2007; Levitin et al., 2008; Coimbra et al., 2009; Li et al., 2010; MacMillan et al., 2010).Conditioned media from in vitro embryogenic cultures contain factors that can promote somatic embryogenesis (SE), implying the presence of secreted signaling molecules (de Vries et al., 1988). There is evidence that secreted AGPs, which are components of conditioned media, are involved in SE. For example, SE in carrot (Daucus carota) and spruce (Picea abies) cell cultures was promoted when AGPs from conditioned media were added exogenously (Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995). Subsequent studies showed the association of particular AGP epitopes with SE-promoting activity and the involvement of AGPs in SE for several other species (Kreuger et al., 1995; McCabe et al., 1997; Toonen et al., 1997; Chapman et al., 2000; Saare-Surminski et al., 2000; Ben Amar et al., 2007). There is also evidence that SE-promoting AGPs may be cleaved by an endochitinase (Egertsdotter and von Arnold, 1988; Domon et al., 2000; van Hengel et al., 2001, 2002), but neither the identity of the individual AGP(s) involved in promoting SE nor the mechanism of action has been established.In this study, we focused on SE in cotton (Gossypium hirsutum ‘Coker 315’), which is a limiting step in cotton transformation, and the potential role of AGPs in this process. We show that cotton calli undergoing somatic embryogenesis secrete an AGP fraction that promotes SE when incorporated back into the growth medium. We report the cloning and sequencing of a complementary DNA (cDNA) encoding a chimeric AGP present in this fraction and show that this molecule promotes SE.