Non-Targeted Metabolite Profiling Reveals Host Metabolomic Reprogramming during the Interaction of Black Pepper with Phytophthora capsici

Phytophthora capsici is one of the most destructive pathogens causing quick wilt (foot rot) disease in black pepper (Piper nigrum L.) to which no effective resistance has been defined. To better understand the P. nigrum–P. capsici pathosystem, we employed metabolomic approaches based on flow‐infusion electrospray–high‐resolution mass spectrometry. Changes in the leaf metabolome were assessed in infected and systemic tissues at 24 and 48 hpi. Principal Component Analysis of the derived data indicated that the infected leaves showed a rapid metabolic response by 24 hpi whereas the systemic leaves took 48 hpi to respond to the infection. The major sources of variations between infected leaf and systemic leaf were identified, and enrichment pathway analysis indicated, major shifts in amino acid, tricarboxylic acid cycle, nucleotide and vitamin B6 metabolism upon infection. Moreover, the individual metabolites involved in defensive phytohormone signalling were identified. RT‐qPCR analysis of key salicylate and jasmonate biosynthetic genes indicated a transient reduction of expression at 24 hpi but this increased subsequently. Exogenous application of jasmonate and salicylate reduced P. capsici disease symptoms, but this effect was suppressed with the co‐application of abscisic acid. The results are consistent with abscisic acid reprogramming, salicylate and jasmonate defences in infected leaves to facilitate the formation of disease. The augmentation of salicylate and jasmonate defences could represent an approach through which quick wilt disease could be controlled in black pepper

Over-expression of bael quinolone synthase in tobacco improves plant vigor under favorable conditions, drought, or salt stress

AbstractType III polyketide synthases (PKSs) catalyze the biosynthesis of various medicinally important secondary metabolites in plants, but their role in growth and stress response is unclear. Here, we overexpressed quinolone synthase (QNS) from bael in tobacco. QNS-overexpressing plants showed an overall increase in growth, photosynthetic efficiency and chlorophyll content compared to wild type plants. Second-generation (T2) transgenic plants grew to maturity, flowered early and set viable seeds under favorable conditions without yield penalty. An increased accumulation of flavonoids, phenols and alkaloids was associated with higher tolerance to drought and salinity stress in transgenic plants. Thus, bael QNS seems to function as a positive regulator of plant growth and stress response, and could be potentially used for engineering plants tolerant to abiotic stress

Amino acid sequence alignment and phylogenetic tree of ginger CDPK1.

<p>(A) The amino acid sequence alignment of <i>Zingiber</i> CDPK1 [GenBank ID: KC544003] with <i>Datura</i> CDPK, DmCPK1 [GenBank ID: 163658596] and <i>Arabidopsis</i> CDPK, AtCPK30 [GenBank ID: 30699042]. Identical amino acids are indicated by asterisks, and similar amino acids are marked with single dots and colons. Catalytic domains (I–XI), Junction domain, and EF hand loops of CaM-LD domain of ZoCDPK1 are marked. The 15 invariant amino acid residues for eukaryotic Ser/Thr protein kinase were highlighted. Protein kinase ATP-binding site is shown by broken lines. Active site is shown in red box. (B) Phylogenetic analysis of ZoCDPK1 with all <i>Arabidopsis</i> CDPKs in the database. The tree was constructed using NJ method of Mega4 software. The numbers on branches showed bootstrap probabilities determined for 1000 re-samplings. The database accession numbers are indicated in parantheses after CDPK gene names.</p

Quantitative real time PCR analysis of <i>ZoCDPK1</i> transcripts in ginger.

<p>(A) <i>ZoCDPK1</i> expression was studied in leaf, stem and rhizome. (B) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post salinity (400 mM NaCl). (C) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post drought (dehydration) treatment. (D) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post ABA (100 μM). (E) Total RNA was isolated from leaves collected at successive intervals of 0, 3, 6, 9, 12, 15 and 24 h post low temperature (4°C) treatment. (F) A time course expression profile of <i>ZoCDPK1</i> against JA (100 µM) was studied at regular intervals of 0, 24, 48, 72 and 96 h post treatment. The <i>EF1α</i> gene was used as endogenous control in all experiments and relative gene expression was calculated by the equation 2^-ddCt. Data are presented as mean <u>+</u> SE (n=3) and error bars represent SE.</p

CDPK1 from Ginger Promotes Salinity and Drought Stress Tolerance without Yield Penalty by Improving Growth and Photosynthesis in <i>Nicotiana tabacum</i>

<div><p>In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) – PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. <i>ZoCDPK1</i> is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The <i>ZoCDPK1</i> up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.</p> </div

Leaf disk senescence assay for salinity and drought tolerance in transgenic tobacco plants (T3).

<p>(A) Representative pictures to show phenotypic differences in leaf disks of wild type (WT), vector control (VC) and T3 plants (CD-1 and CD-6) after incubation in 0, 100, 200, 300, 400 and 500 mM NaCl or 500 mM mannitol solutions. (B) Representative diagram to show the chlorophyll content from leaf disks of wild type, vector control and T3 transgenic plants after incubation in 200 mM NaCl or 300 mM mannitol solutions. Data are presented as mean <u>+</u> SE (n=3) and error bars represent SE. Data shows a significant difference of chl.a or chl.b content between transgenic and wild type controls at P<0.0001, by Student’s t-test. </p

Analysis of ZoCDPK1 transgenic (T3) plants under salinity and drought.

<p>(A) Representative pictures to show the germination of transgenic (CD-1 and CD-6), vector control (VC) and wild type (WT) seeds under normal conditions (non-stress), 200 mM NaCl (salinity) and 300 mM mannitol (drought). (B) Representative seedlings of WT, VC and T3 homozygous lines (CD-1 and CD-6) taken after 14 days of germination on normal (non-stress), 200 mM NaCl (Upper) and 300 mM mannitol (Lower) conditions.</p