Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Summary

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Abstract

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.

Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.

In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Introduction

Plants produce a wide huge variety of compounds with different industrial and pharmaceutical applications, ranging from small molecular weight molecules to polymers. Such compounds are frequently produced and secreted by highly specialized structures, many of which are localized at the surface of the plants such as glandular trichomes or nectaries, or internally such as idioblasts or latex and resin ducts, respectively. However, relatively little is known about the biology of these specialized secretory structures, although all types of those glands display many features that are indicative of active metabolism. In particular, glands of carnivorous plants such as ....

Protocol

1. Plant Material Preparation

1. All Nepenthes species were grown either in the greenhouses of the MPI for Chemical Ecology or the Botanical Garden in Jena, Germany.
2. For the preparation of secreting tissue harvest a pitcher and remove the upper one third including lid, peristome, and slippery zone. The pitcher can be used either freshly or frozen in liquid nitrogen and stored at -80 °C.

2. Microdissection of Pitcher Tissue

1. The microsampling platform consists of a stereomicroscope and a micromanipulator, which is directly attached to the microscope body and is operated by a 3D joystick. Her....

Results

For the preparation of single glands from Nepenthes pitcher tissue, a mechanized microdissection technique was developed and applied. The micropreparation platform needs to be equipped with microforceps, attached to the micromanipulator, which in turn are operated by a control panel. Individual glands, visible under the microscope, could be targeted with the forceps that grasped the gland tissue and removed it mechanically (Figure 4A).

The glandular tissue was transfe.......

Discussion

For a long time, studies of plant secretory structures, in particular trichomes have been performed essentially on anatomy and were purely descriptive. There is a large body of publications of this type as summarized in reviews by^9-11. Our biochemical and molecular knowledge of plant secretory structures is still limited in spite of some early remarkable work e.g. on mint glandular trichomes showing that glandular trichomes are the site of biosynthesis of monoterpenoids which are secreted into the sub.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNAqueous-Micro Kit	Life Technologies GmbH, Germany	AM1931	Isolation of total RNA, 50 samples
Dynabeads mRNA DIRECT Micro Kit	Life Technologies GmbH, Germany	61011	5 ml Dynabeads Oligo (dT)25
DynaMag-2 magnet	Life Technologies GmbH, Germany	12321D
SuperScript CellsDirect cDNA Synthesis System	Life Technologies GmbH, Germany	18080-200	25 reactions
RNaseOUT	Life Technologies GmbH, Germany	10777-019	Rnase inhibitor, 5,000 units
Random Hexamers (0.4 μg/μl)	QIAGEN GmbH, Germany	79236	100 μl
10 mM dNTP Mix, molecular biology grade	Fermentas GmbH, Germany	R0192	1 ml
Taq DNA polymerase, (recombinant)	Fermentas GmbH, Germany	EP0402	Supplied with 10x Taq buffer (with KCl or (NH4)2SO4) and 25 mM MgCl2
TURBO DNase	Life Technologies GmbH, Germany	AM2238	1,000 units
Agarose	Carl Roth GmbH Co. Germany	T846.3
GeneRuler Low Range DNA ladder, ready-to-use	Fermentas GmbH, Germany	SM1193	50 μg
Brilliant II SYBR Green QPCR Master Mix	Agilent Technologies, Inc., USA	600828	Single kit
RNaseZAP	Sigma-Aldrich, Taufkirchen, Germany	R2020
Razor blade	Carl Roth GmbH Co., Germany	CK08.1
Tweezers	Carl Roth GmbH Co., Germany	2801.1 / 2855.1
GE NanoVue spectrophotometer	GE Healthcare Europe GmbH, Germany	28-9569-62
Mastercycler gradient	Eppendorf AG, Hamburg, Germany	5331 000.010
Mx3000P Real-Time PCR System	Stratagene, USA	401403
Agagel Standard Horizontal Gel Electrophoresis Chamber	Biometra	Discontinued
Micromanipulator	aura optik gmbh	aureka	Equipped with a microforceps
Stereomicroscope	Carl Zeiss Microscopy, Jena, Germany	SteREO Lumar.V12	Equipped with an AxioCam
Lumar Filter set 01	Carl Zeiss, Microscopy, Jena, Germany	485001-0000-000	UV filter
Lumar Filter set 09	Carl Zeiss, Microscopy, Jena, Germany	485009-0000-000	non-UV filter