Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants.

PREMISE:A portable, simple, yet efficient method was developed for the rapid extraction of xylem sap from the stems and petioles of tomato plants for diagnostic and quantification assays of the xylem-colonizing wilt bacterium Ralstonia solanacearum. METHODS AND RESULTS: Xylem saps were extracted from tomato stem sections using negative pressure generated from handheld needleless syringes. The samples were collected from plants grown under different soil moisture levels at four days after inoculation with the pathogen. Pipette tips were modified to serve as adapters for the stem sections. The quantification of the bacterial load in the extracted sap was performed by plating sap dilutions in Kelman's triphenyltetrazolium chloride (TTC) medium. Pathogen identity was further confirmed by performing a PCR using R. solanacearum-specific primers.CONCLUSIONS: Due to its simplicity, portability, and thoroughness of extraction from predetermined tissue sizes, the method can potentially facilitate high-throughput onsite sampling from a large number of samples in a short time, which cannot be achieved with other available techniques. KEY WORDS negative pressure; Ralstonia solanacearum; tomato stem; xylem sap extraction. Applications in Plant Sciences 2020 8(4): e11335 Longchar et al.-Stem xylem sap extraction • 2 of 8

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Biosynthesis of sterols is a multistep process in higher plants where the precursor cycloartenol gets converted into functional phytosterols after removal of two methyl groups at C-4 by an enzyme complex involving a sterol C-4 methyl oxidase (SMO). We identified and cloned a cDNA from Artemisia annua designated as AaSMO1 showing similarity to SMO. The cDNA predicted to encode a polytopic protein with characteristic histidinerich motifs and an ER retrieval signal. GFP-AaSMO1 fusion protein was localized in endoplasmic reticulum of transformed protoplast and onion epidermal cells. AaSMO1 expression was drastically induced upon osmotic/dehydration stress and its promoter showed the presence of abscisic acid responsive element. Transgenic tobacco plants ectopically overexpressing AaSMO1 were raised, and various biochemical and physiological analyses of transgenics revealed increased total sterol, better germination and growth in subsequent generations. They also exhibited reduced sensitivity towards osmotic/dehydration stress which may be attributed to enhanced SMO1 activity. Our studies demonstrated that apart from acting as phytohormones, plant sterols also participate in providing capability to plants for improved growth and adaptation during stress conditions. AaSMO1 can be used as an excellent candidate for generating dehydration/drought tolerant plants.

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