Plant Science 151 2000 75 – 83
Identification of new nodulin cDNAs from yellow lupine by differential display
Michal R. Swiderski , Zaneta Zaborowska, Andrzej B. Legocki
Institute of Bioorganic Chemistry PAN, Noskowskiego
12 14
,
61
-
704
Poznan, Poland Received 2 June 1999; received in revised form 18 August 1999; accepted 28 September 1999
Abstract
In this study several sequences that represent yellow lupine nodulin genes are identified. Four different cDNA clones were isolated from a lupine nodule cDNA library and sequenced. Database search revealed that the deduced amino acid of one clone,
DD3A20, shows near 80 amino acid similarity to plant stearoyl-acyl carrier protein desaturases and contains crucial amino acids in the fatty acid binding domain which are characteristic for stearoyl-ACP desaturases. The other sequences seem to be unique.
Nearly identical signal peptide sequences are present at the N-termini of the DD4A9 and DD2T15 putative proteins. Northern analysis indicated that the pattern of expression of the Lldd
4
A
9
, Lldd
2
A
18
and Lldd
2
T
15
genes is the same as that of the lupine leghemoglobin gene LllbII, while the desaturase gene Ll
3
A
20
is turned on at least 3 days earlier. Ll
3
A
20
is also expressed in lupine fruits. RT-PCR analysis of the Lldd
3
A
20
and Lldd
2
T
15
expression confirmed organ-specific and temporal pattern of expression in nodules. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Yellow lupine; Nodules; Nodulins; Desaturase www.elsevier.comlocateplantsci
1. Introduction
Infection of legume plants with specific strains of
Rhizobium, Bradyrhizobium,
Azorhizobium, Sinothizobium or Mesorhizobium leads to the de-
velopment of specialized nitrogen fixing organs called root nodules. Although nodulation is con-
trolled by genetic determinants of both symbionts, the type of root nodule depends on the plant
species [1]. There are two major classes of nodules that are formed on legume roots. Indeterminate
nodules usually develop on temperate legumes. These nodules are characterized by the presence of
a persistent apical meristem, with initial cell divi- sions occurring in the deeper cell layers of cortex,
close to endodermis [1 – 3]. Growth and differenti- ation of new cells leads to the establishment of
central tissue zonation. Four zones have been distinguished in mature alfalfa nodules: bacterium-
free meristematic zone I; infection zone II; nitro- gen fixing zone III and senescent zone IV [4].
Determinate type nodules are characteristic for tropical legumes such as Glycinae, Phaseolus or
Vigna. In this class of nodules meristematic activ- ity ceases after several days of development and
subsequent growth is caused by cell expansion. The first cell divisions are hypodermic and in
mature nodules the central tissue does not show zonation [1 – 3].
Nodulins were, by the original definition, plant gene products appearing exclusively during nodule
formation [5]. However, the term nodulin gene is used in a less strict sense and refers to genes
Corresponding author. Present address: Department of Biology, Indiana University, Bloomington, IN 47405, USA. Tel.: + 1-812-855-
2852; fax: + 1-812-855-6705. E-mail address
:
mswidersbio.indiana.edu M.R. Swiderski 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 9 8 - 3
expressed in nodules, but not in uninfected roots. Intensive studies over the past two decades have
identified almost 200 nodulin genes in different plant hosts [6]. Nodulins are defined as early or
late according to the time of appearance during nodule development. Early nodulins are expressed
well before nitrogen fixation onset and function in nodule development and infection processes [3,5].
Although the expression of several early nodulin genes has been well documented, the exact roles of
the encoded proteins are still unknown [7 – 11]. Late nodulins participate in nodule function and
appear in nodules during the onset of nitrogen fixation, after the nodule structure has developed
[3,5]. This group of nodulins includes leghemo- globins; enzymes functioning in nitrogen and car-
bon metabolism glutamine synthetase, uricase, sacharose synthatese; peribacteroid membrane
proteins and many others, of which the function remains to be elucidated [3,12 – 16]. Recently, it
has
been reported
that the
Vicia sati6a
leghemoglobin gene Vslb
1
, is expressed in root hairs within 1 h after Nod factor treatment indi-
cating that the function of some nodulins may have to be revised [17].
Lupine root nodules have been described as a unique lupinoid subtype within the indeterminate
class [18]. The meristem is located basi-laterally and contains bacteroids, so there is no distinctly
marked infection zone. Initial cell divisions occur hypodermally. During nodule growth the connec-
tion to the root broadens causing the nodule to surround the root [18].
Several lupine nodulin genes have been iden- tified and characterized. These include genes cod-
ing for two leghemoglobins LbI and LbII, glutamine synthetase and ENOD2-like protein
[19,20]. To identify additional nodulin genes and to increase the repertoire of usable molecular
probes, the differential display approach was em- ployed [21,22]. Differential display has been suc-
cessfully used to identify new nodulin genes from Sesbania rostrata and to prepare a nodulin ex-
pressed sequence tag library from Lotus japonicus [23,24]. In this study expression analysis of several
newly identified nodulin genes during lupine nod- ule development is presented. One of the isolated
clones encodes a putative stearoyl-acyl carrier protein desaturase. A function for this enzyme in
nodule development is proposed.
2. Material and methods
