Dual Activities of ACC Synthase: Novel Clues Regarding the Molecular Evolution of Acs Genes

Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of S-adenosyl-methionine to the ethylene precursor ACC, genuine ACSs widely have Cβ-S lyase activity. Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes

Cytokinin-induced gene expression in Arabidopsis

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of Arabidopsis thaliana Landsberg erecta (L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants

PROSYSTEMIN: A HUB OF TOMATO PLANT DEFENSE RESPONSES

Plants live in a complex environment suffering various stress constraints. To counteract stress condition plants have evolved sophisticated defense systems. In tomato plants a key role in defense is played by systemin (Sys), an octadecapeptide, released upon leaf damage from a larger precursor, prosystemin (Prosys). Considering the need to reduce the agro-chemicals we investigated foliar and hydroponic application of Sys to tomato plants that increased both direct and indirect defenses (Chapter 1): treated plants strongly reduce growth and vitality of Spodoptera littoralis larvae also damaging the development of future insect generations. In addition, Sys treated plants reduce leaves colonization of the necrotrophic fungus Botrytis cinerea and have an increased level of attractiveness of natural herbivores antagonists. In order to investigate the molecular mechanism underpinning Prosys' defence activation, a prediction study of protein-protein interactions (PPIs) was done (Chapter 2). More than 16000 interactions were captured from the interactome query and, among them, 98 Prosys direct interactors were catalogued using GO terms. Prosys sub-network evidenced that Prosys links with two large groups of kinases and transcription factors confirming that the precursor is associated with the very early steps of plant stress perception. Prosys PPIs were also investigated in vitro and in vivo (Chapter 3). Affinity Purification Mass Spectrometry (AP-MS) detected more than 300 Prosys interactors, including two molecular partners identified in silico, a heat shock protein 70 (Sl-HSP70-1), which plays a key role in stress responses, and NAD-dependent epimerase\dehydratase (NaDED), likely associated with both sugar and hormonal plant defense signalling. Some PPIs were validated through BiFC that confirmed the interaction with an ATP-dependent clp protease, detected with AP-MS, and with the NaDED, detected both in silico and in vitro. BiFC also confirmed two interactors of the in silico network, MYB transcription factor and a MAP-Kinase. Overall, the results proved that Sys is a very effective plant protectant, and its use could reduce the application of chemical pesticide while Prosys is involved in a large number of interactions possibly due to its ID structure and consequent biological function

Dissecting regulatory eQTLs of the carotenoid biosynthetic pathway in tomato fruit using S.lycopersicum x S. pennellii introgression lines

Tomato (Solanum lycopersicum) ripening involves a number of physiological processes that include the visible breakdown of chlorophyll and build-up of carotenoids, with massive accumulation of antioxidant compounds such as lycopene and β-carotene (provitamin A) within the chromoplasts. Although the catalytic steps of the carotenoid biosynthetic pathway have been well-characterised, the regulatory mechanisms that control carotenoid accumulation remain poorly understood. In this study, I used expression quantitative trait loci (eQTL) analysis to obtain a comprehensive understanding of the genetic basis of regulation of carotenoid biosynthesis in tomato fruit using the S. lycopersicum x S. pennellii introgression population. In total, 31 cis-eQTLs related to 18 carotenoid biosynthetic genes were identified, and the isoforms of some structural genes functional during fruit ripening were identified in this analysis. Six trans-eQTL hotspots were identified for lycopene biosynthesis. Co-expression analysis of one of the trans-eQTL candidates, residing in IL2-1, revealed that a basic helix-loop-helix transcription factor, SlHONG likely acts as a positive regulator of lycopene accumulation in fruit, by activating the expression of the structural genes involved in lycopene biosynthesis. SlRIN, a master regulator of fruit ripening, binds directly to two adjacent CArG motifs in the promoter of SlHONG, to control the expression of SlHONG in an ethylene-independent manner. Genome-scale phylogenetic analysis of bHLH proteins in Arabidopsis and tomato found SlHONG was the most similar protein to BEE2 in Arabidopsis, which is involved in brassinosteroid signalling by forming the BEE complex together with another three bHLH transcription factors. bHLH transcription factors potentially involved in the BEE complex with SlHONG in tomato, were identified by phylogenetic analysis and interactions between the candidate proteins were detected. SlHONG was identified as a regulator of lycopene biosynthesis in tomato fruit, and may be under the control of multi-layered regulatory mechanisms

Deciphering the Indoleacetic Acid Biosynthesis Pathways in the Rhizobacterium Pseudomonas sp. UW4

Healthy plants host, within and on the surfaces of their tissues, diverse endophytic and epiphytic bacteria. Often, this interaction is mutualistic, leading to adaptive benefits for both partners. We refer to these beneficial microbes as plant growth-promoting bacteria (PGPB), as they can have a tremendous positive influence on plant health, yield and productivity. PGPBs can be used as natural biofertilizers to promote plant growth in an environmentally responsible manner. One of the main mechanisms used by PGPB to enhance plant growth is the production of indoleacetic acid (IAA). This compound is central to a plant’s lifecycle and overall functioning. Because of the indispensable role of IAA in plant-growth promotion, there is great interest in genetic manipulation of IAA biosynthesis to maximize phytostimulation. This is a cumbersome task, as the nature of IAA biosynthesis is convoluted; multiple, independent and inter-dependent pathways operate within a single bacterium. The research reported herein strived to decipher the IAA biosynthesis pathways at the genetic and biochemical level, in a particularly effective PGPB known as Pseudomonas sp. UW4. This remarkable rhizobacterium has been shown to enhance plant growth in the presence of flooding, heavy metals, cold, high levels of salt, and phytopathogens. The entire genome of strain UW4 was sequenced in our lab and seven genes were implicated to encode enzymes involved in the indole acetonitrile (IAN) and indoleacetamide (IAM) pathways of IAA biosynthesis. In this work, some of these enzymes were isolated and their catalytic activity was experimentally verified through biochemical assays. Transformants of strain UW4 with increased IAA biosynthetic capacity were created by introducing a second copy of the target IAA-genes, and canola seedlings inoculated with these transformants displayed enhanced root growth. Mutagenesis experiments were undertaken to create deletions in all seven IAA-genes, in order to delineate which of the genes/pathways contribute most to IAA production in this strain. Failure to produce mutants with a reduced ability to synthesize IAA, led to speculation of a third pathway. Biochemical evidence of this pathway is based on the detection of the IPyA metabolite. The IPyA pathway appears to be predominant in strain UW4 and can compensate for disruptions in the IAN/IAM pathways. Genomic screens identified several candidate IPyA pathway genes, however the functional roles of the encoded enzymes remain to be determined. Altogether, this work describes three interconnected IAA biosynthetic pathways in strain UW4

Function of hybrid histidine kinases in Arabidopsis flagellin-mediated defence responses

In plants, the first line of microbial recognition relies on the perception of pathogen-associated molecular patterns (PAMPs) allowing plants to detect microorganisms and respond with a set of basal defence responses. The best studied PAMP is flagellin, the main protein component of bacterial flagella. The sensor histidine kinase AHK5 has been shown to play a novel role in mediating flagellin-induced stomatal closure. AHK5 belongs to a family of 9 Arabidopsis hybrid-histidine kinases (HKs). To further investigate the role of such HKs in flagellin-induced signal transduction, physiological responses to the flagellin derived peptide flg22 were examined in available hybrid HK mutant lines. Seedlings of the ethylene insensitive HK ETR1 mutant (etr1-1) showed dramatically reduced flg22 sensitivity as assayed by flg22-mediated seedling growth inhibition. A novel role for the hormone ethylene in flg22-mediated growth inhibition was thus identified. Conversely enhanced sensitivity to low concentrations of flg22 was observed in the AHK2 cytokinin receptor mutant (ahk2-2). However, the absence of flg22-associated growth phenotype in other cytokinin receptor mutants would suggest the role of AHK2 in flg22-mediated seedling growth inhibition may be independent of its role in cytokinin perception. Despite a wild-type sensitivity in aerial plant tissues, distinct flg22-mediated root growth arrest phenotypes were observed in plants defective in the HKs ETR1 and AHK5. Dissection of the mechanisms underlying flg22-mediated root growth inhibition led to the identification of nitric oxide and the ethylene precursor ACC as key secondary messengers. Further characterisation of etr1 mutants showed that, in addition to seedling growth inhibition, ethylene perception is also required for flg22-mediated callose deposition however surprisingly, does not appear to be a requirement for flg22-mediated bacterial immunity. Despite the known requirement for AHK5 in flg22-mediated stomatal closure, flg22-mediated post-invasive bacterial defences were found to be intact in ahk5-1 mutant plants. In summary this study has shown that ethylene perception via the ethylene receptor HK family plays an integral part in flg22-mediated signalling. In addition, organ/tissue specific functions for three of the nine hybrid kinases, AHK2, AHK5 and ETR1 in flg22-mediated signal transduction have been identified