Project Faria

Molecular, cellular and physiological aspects of desiccation sensitivity in Medicago truncatula and Inga vera seeds

José Faria (Ph.D student Plant Cell Biology and Plant Physiology)
Dr. André van Lammeren (supervisor)

Dr. Henk Hilhorst (supervisor Laboratory of Plant Physiology)

During the maturation drying phase of their development, orthodox seeds display a developmental arrest (DA), with the purportedly involvement of several genes (e.g. ABI3, LEC1 and FUS3) and a crucial role of abscissic acid (ABA). DA leads seeds to a quiescent state, enabling them to withstand further desiccation to very low moisture contents (MCs) and long-term storage. With opposite behavior, recalcitrant seeds generally proceed directly from developmental to germinative program, without interruption by DA. These seeds, shed metabolically active and with high MCs, are sensitive to desiccation, making their storage very difficult. In order to warrant long term storage of recalcitrant seeds, e.g. for seed bank purposes, it is important to know the causes of desiccation sensitivity in seeds. The present study focuses on two major directions: 1) cell cycle (DNA content and the microtubule cytoskeleton) and 2) desiccation-related and stress-inducible genes. The investigation is being performed on two species: Inga vera (a legume tree species native to Brazil, whose seeds are extremely desiccation sensitive) and the model species Medicago truncatula, also a legume, whose seeds are desiccation tolerant. To study desiccation sensitivity in M. truncatula seeds, the stage during which desiccation tolerance (DT) is lost, namely the moment of visible germination (radicle protrusion) is studied. Recently a method has been developed that allows the re-establishment of DT in germinated seeds of M. truncatula, by treating them with polyethylene glycol (PEG), generating a new tool for studies on desiccation (in)tolerance in seeds. This method has been used successfully in the present study.

faria02.jpg

http://rsta.pucmm.edu.do/ciencias/arboretum/intro/website%20sub%20pages/Arboretum/Area_3/area_3-c/8-3c/body_8-3c.html

faria04.jpg

http://www.fao.org/ag/AGP/AGPC/doc/gallery/pictures/meditrunc/meditrunc.htm

The questions addressed can be summarised as: (1) How does desiccation sensitivity in I.vera seeds and in germinating M. truncatula seeds relate to the progress of the cell cycle? (2) Is ABI3 (and related genes) absent in developing I. vera seeds and does this explain the absence of DA and desiccation tolerance? (3) Does the expression of ABI3 (and related genes) cease in germinating M. truncatula seeds and does this explain the loss of DT? (4) Is the expression of ABI3 (and related genes) linked to the presence of ABA? (5) Does the expression of ABI3 (and related genes) resume in germinating M. truncatula seeds when treated with PEG? Does this indicate a 'return' to the developmental program? (6) What is the feasibility of applying such PEG treatment in I. vera seeds in order to diminish their desiccation sensitivity and improve their storability? (7) Which stress-inducible genes (e.g. LEA genes) are expressed in I. vera seeds and in germinating M. truncatula seeds when treated with PEG?

To elucidate these questions, several approaches are being (or will be) used, such as DNA content assessment (by flow cytometry); immunohistochemical detection of microtubules and tubulin; ABA quantification and study of gene expression (using RT-PCR, Northern blotting, in situ hybridization and DNA micro-array).