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<jats:p>The frequency and intensity of drought events are predicted to increase because of climate change, threatening biodiversity and terrestrial ecosystems in many parts of the world. Drought has already led to declines in functionally important tree species, which are documented in dieback events, shifts in species distributions, local extinctions, and compromised ecosystem function. Understanding whether tree species possess the capacity to adapt to future drought conditions is a major conservation challenge. In this study, we assess the capacity of a functionally important plant species from south-eastern Australia (<jats:italic>Banksia marginata</jats:italic>, Proteaceae) to adapt to water-limited environments. A water-manipulated common garden experiment was used to test for phenotypic plasticity and genetic adaptation in seedlings sourced from seven provenances of contrasting climate-origins (wet and dry). We found evidence of local adaptation relating to plant growth investment strategies with populations from drier climate-origins showing greater growth in well-watered conditions. The results also revealed that environment drives variation in physiological (stomatal conductance, predawn and midday water potential) and structural traits (wood density, leaf dry matter content). Finally, these results indicate that traits are coordinated to optimize conservation of water under water-limited conditions and that trait coordination (phenotypic integration) does not constrain phenotypic plasticity. Overall, this study provides evidence for adaptive capacity relating to drought conditions in <jats:italic>B. marginata</jats:italic>, and a basis for predicting the response to climate change in this functionally important plant species.</jats:p>

<jats:sec><jats:title>Introduction</jats:title><jats:p><jats:italic>Trans</jats:italic>-cinnamaldehyde is a specialised metabolite that naturally occurs in plants of the Lauraceae family. This study focused on the phytotoxic effects of this compound on the morphology and metabolism of <jats:italic>Arabidopsis thaliana</jats:italic> seedlings.</jats:p></jats:sec><jats:sec><jats:title>Material and methods</jats:title><jats:p>To evaluate the phytotoxicity of <jats:italic>trans</jats:italic>-cinnamaldehyde, a dose-response curve was first performed for the root growth process in order to calculate the reference inhibitory concentrations IC50 and IC80 (<jats:italic>trans</jats:italic>-cinnamaldehyde concentrations inducing a 50% and 80% inhibition, respectively). Subsequently, the structure and ultrastructure of the roots treated with the compound were analysed by light and electron microscopy. Based on these results, the following assays were carried out to in depth study the possible mode of action of the compound: antiauxinic PCIB reversion bioassay, determination of mitochondrial membrane potential, ROS detection, lipid peroxidation content, hormone quantification, <jats:italic>in silico</jats:italic> studies and gene expression of ALDH enzymes.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p><jats:italic>Trans</jats:italic>-cinnamaldehyde IC50 and IC80 values were as low as 46 and 87 μM, reducing the root growth and inducing the occurrence of adventitious roots. At the ultrastructural level, the compound caused alterations to the mitochondria, which were confirmed by detection of the mitochondrial membrane potential. The morphology observed after the treatment (i.e., appearance of adventitious roots) suggested a possible hormonal mismatch at the auxin level, which was confirmed after PCIB bioassay and hormone quantification by GC-MS. The addition of the compound caused an increase in benzoic, salicylic and indoleacetic acid content, which was related to the increased gene expression of the aldehyde dehydrogenase enzymes that can drive the conversion of <jats:italic>trans</jats:italic>-cinnamaldehyde to cinnamic acid. Also, an increase of ROS was also observed in treated roots. The enzyme-compound interaction was shown to be stable over time by docking and molecular dynamics assays.</jats:p></jats:sec><jats:sec><jats:title>Discussion</jats:title><jats:p>The aldehyde dehydrogenases could drive the conversion of <jats:italic>trans</jats:italic>-cinnamaldehyde to cinnamic acid, increasing the levels of benzoic, salicylic and indoleacetic acids and causing the oxidative stress symptoms observed in the treated seedlings. This would result into growth and development inhibition of the <jats:italic>trans</jats:italic>-cinnamaldehyde-treated seedlings and ultimately in their programmed-cell-death.</jats:p></jats:sec>

<jats:p>Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type <jats:italic>Arabidopsis</jats:italic> and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.</jats:p>

<jats:p>As a conspicuous trait, peel color is one of the most important characteristics that affects commodity quality and consumer preferences. The locus <jats:italic>Y</jats:italic> underlying yellow peel in <jats:italic>Cucurbita pepo</jats:italic> (zucchini) was first reported in 1922; however, its molecular mechanism is still unknown. In this study, a genetic analysis revealed that yellow peel is controlled by a single dominant genetic factor. Furthermore, <jats:italic>Y</jats:italic> was mapped in a ~170 kb region on chromosome 10 by bulked segregated analysis (BSA) and fine mapping in F<jats:sub>2</jats:sub> and BC<jats:sub>1</jats:sub> segregating populations. The candidate region harbors fifteen annotated genes, among which <jats:italic>Cp4.1LG10g11560</jats:italic> (<jats:italic>CpCHLH</jats:italic>) is regarded as a promising candidate gene. <jats:italic>CpCHLH</jats:italic> encodes a magnesium chelatase H subunit involved in chlorophyll biosynthesis, and its mutation can result in a reduction in chlorophyll content and yellow phenotype. Interestingly, a large fragment (~15 kb) duplication containing incomplete <jats:italic>CpCHLH</jats:italic> was inserted in the candidate interval, resulting in two reformed CpCHLH proteins in the yellow parental line. It is most likely that the reformed CpCHLH proteins act as a malfunctional competitor of the normal CpCHLH protein to interrupt the formation of chlorophyll. Overall, the isolation of <jats:italic>Y</jats:italic> will shed light on the molecular mechanism of the peel color regulation of zucchini and lay a foundation for breeding.</jats:p>

<jats:p>Nitrogen (N) is an essential macronutrient for plants, acting as a common limiting factor for crop yield. The application of nitrogen fertilizer is related to the sustainable development of both crops and the environment. To further explore the molecular response of sugar beet under low nitrogen (LN) supply, transcriptome analysis was performed on the LN-tolerant germplasm ‘780016B/12 superior’. In total, 580 differentially expressed genes (DEGs) were identified in leaves, and 1,075 DEGs were identified in roots (log<jats:sub>2</jats:sub><jats:sup>|FC|</jats:sup> ≥ 1; q value &amp;lt; 0.05). Gene Ontology (GO), protein−protein interaction (PPI), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses clarified the role and relationship of DEGs under LN stress. Most of the downregulated DEGs were closely related to “photosynthesis” and the metabolism of “photosynthesis-antenna proteins”, “carbon”, “nitrogen”, and “glutathione”, while the upregulated DEGs were involved in flavonoid and phenylalanine biosynthesis. For example, <jats:italic>GLUDB</jats:italic> (glutamate dehydrogenase B) was identified as a key downregulated gene, linking carbon, nitrogen, and glutamate metabolism. Thus, low nitrogen-tolerant sugar beet reduced energy expenditure mainly by reducing the synthesis of energy-consuming amino acids, which in turn improved tolerance to low nitrogen stress. The glutathione metabolism biosynthesis pathway was promoted to quench reactive oxygen species (ROS) and protect cells from oxidative damage. The expression levels of nitrogen assimilation and amino acid transport genes, such as <jats:italic>NRT2.5 </jats:italic> (high-affinity nitrate transporter), <jats:italic>NR</jats:italic> (nitrate reductase [NADH]), <jats:italic>NIR</jats:italic> (ferredoxin-nitrite reductase), <jats:italic>GS</jats:italic> (glutamine synthetase leaf isozyme), <jats:italic>GLUDB, GST</jats:italic> (glutathione transferase) and <jats:italic>GGT3</jats:italic> (glutathione hydrolase 3) at low nitrogen levels play a decisive role in nitrogen utilization and may affect the conversion of the carbon skeleton. <jats:italic>DFRA</jats:italic> (dihydroflavonol 4-reductase) in roots was negatively correlated with <jats:italic>NIR</jats:italic> in leaves (coefficient = −0.98, p &amp;lt; 0.05), suggesting that there may be corresponding remote regulation between “flavonoid biosynthesis” and “nitrogen metabolism” in roots and leaves. <jats:italic>FBP</jats:italic> (fructose 1,6-bisphosphatase) and <jats:italic>PGK</jats:italic> (phosphoglycerate kinase) were significantly positively correlated (p &amp;lt; 0.001) with Ci (intercellular CO<jats:sub>2</jats:sub> concentration). The reliability and reproducibility of the RNA-seq data were further confirmed by real-time fluorescence quantitative PCR (qRT−PCR) validation of 22 genes (R<jats:sup>2</jats:sup> = 0.98). This study reveals possible pivotal genes and metabolic pathways for sugar beet adaptation to nitrogen-deficient environments.</jats:p>

<jats:p>Shikonin derivatives are natural naphthoquinone compounds and the main bioactive components produced by several boraginaceous plants, such as <jats:italic>Lithospermum erythrorhizon</jats:italic> and <jats:italic>Arnebia euchroma</jats:italic>. Phytochemical studies utilizing both <jats:italic>L. erythrorhizon</jats:italic> and <jats:italic>A. euchroma</jats:italic> cultured cells indicate the existence of a competing route branching out from the shikonin biosynthetic pathway to shikonofuran. A previous study has shown that the branch point is the transformation from (<jats:italic>Z</jats:italic>)-3’’-hydroxy-geranylhydroquinone to an aldehyde intermediate (<jats:italic>E</jats:italic>)-3’’-oxo-geranylhydroquinone. However, the gene encoding the oxidoreductase that catalyzes the branch reaction remains unidentified. In this study, we discovered a candidate gene belonging to the cinnamyl alcohol dehydrogenase family, <jats:italic>AeHGO</jats:italic>, through coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines of <jats:italic>A. euchroma</jats:italic>. In biochemical assays, purified AeHGO protein reversibly oxidized (<jats:italic>Z</jats:italic>)-3’’-hydroxy-geranylhydroquinone to produce (<jats:italic>E</jats:italic>)-3’’-oxo-geranylhydroquinone followed by reversibly reducing (<jats:italic>E</jats:italic>)-3’’-oxo-geranylhydroquinone to (<jats:italic>E</jats:italic>)-3’’-hydroxy-geranylhydroquinone, resulting in an equilibrium mixture of the three compounds. Time course analysis and kinetic parameters showed that the reduction of (<jats:italic>E</jats:italic>)-3’’-oxo-geranylhydroquinone was stereoselective and efficient in presence of NADPH, which determined that the overall reaction proceeded from (<jats:italic>Z</jats:italic>)-3’’-hydroxy-geranylhydroquinone to (<jats:italic>E</jats:italic>)-3’’-hydroxy-geranylhydroquinone. Considering that there is a competition between the accumulation of shikonin and shikonofuran derivatives in cultured plant cells, AeHGO is supposed to play an important role in the metabolic regulation of the shikonin biosynthetic pathway. Characterization of AeHGO should help expedite the development of metabolic engineering and synthetic biology toward production of shikonin derivatives.</jats:p>

<jats:p><jats:italic>Artemisia argyi</jats:italic> (<jats:italic>A. argyi</jats:italic>) is a medicinal plant belonging to the <jats:italic>Asteraceae</jats:italic> family and <jats:italic>Artemisia genus</jats:italic>. Flavonoids abundant in <jats:italic>A. argyi</jats:italic> are associated with anti-inflammatory, anticancer, and antioxidative effects. Eupatilin and jaceosidin are representative polymethoxy flavonoids with medicinal properties significant enough to warrant the development of drugs using their components. However, the biosynthetic pathways and related genes of these compounds have not been fully explored in <jats:italic>A. argyi.</jats:italic> This study comprehensively analyzed the transcriptome data and flavonoids contents from four different tissues of <jats:italic>A. argyi</jats:italic> (young leaves, old leaves, trichomes collected from stems, and stems without trichomes) for the first time. We obtained 41,398 unigenes through the <jats:italic>de-novo</jats:italic> assembly of transcriptome data and mined promising candidate genes involved in the biosynthesis of eupatilin and jaceosidin using differentially expressed genes, hierarchical clustering, phylogenetic tree, and weighted gene co-expression analysis. Our analysis led to the identification of a total of 7,265 DEGs, among which 153 genes were annotated as flavonoid-related genes. In particular, we were able to identify eight putative flavone-6-hydroxylase (<jats:italic>F6H</jats:italic>) genes, which were responsible for providing a methyl group acceptor into flavone basic skeleton. Furthermore, five <jats:italic>O</jats:italic>-methyltransferases (<jats:italic>OMT</jats:italic>s) gene were identified, which were required for the site-specific <jats:italic>O</jats:italic>-methylation during the biosynthesis of eupatilin and jaceosidin. Although further validation would be necessary, our findings pave the way for the modification and mass-production of pharmacologically important polymethoxy flavonoids through genetic engineering and synthetic biological approaches.</jats:p>

<jats:p>Phytohormones play an important role in regulating the plant behavior to drought. In previous studies, NIBER<jats:sup>®</jats:sup> pepper rootstock showed tolerance to drought in terms of production and fruit quality compared to ungrafted plants. In this study, our hypothesis was that short-term exposure to water stress in young, grafted pepper plants would shed light on tolerance to drought in terms of modulation of the hormonal balance. To validate this hypothesis, fresh weight, water use efficiency (WUE) and the main hormone classes were analyzed in self-grafted pepper plants (variety onto variety, V/V) and variety grafted onto NIBER<jats:sup>®</jats:sup> (V/N) at 4, 24, and 48h after severe water stress was induced by PEG addition. After 48h, WUE in V/N was higher than in V/V, due to major stomata closure to maintain water retention in the leaves. This can be explained by the higher abscisic acid (ABA) levels observed in the leaves of V/N plants. Despite the interaction between ABA and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in relation to stomata closure is controversial, we observed an important increase of ACC at the end of the experiment in V/N plants coinciding with an important rise of the WUE and ABA. The maximum concentration of jasmonic acid and salicylic acid after 48h was found in the leaves of V/N, associated with their role in abiotic stress signaling and tolerance. Respect to auxins and cytokinins, the highest concentrations were linked to water stress and NIBER<jats:sup>®</jats:sup>, but this effect did not occur for gibberellins. These results show that hormone balance was affected by water stress and rootstock genotype, where NIBER<jats:sup>®</jats:sup> rootstock displayed a better ability to overcome short-term water stress.</jats:p>