The new xantha-variegata-viredescens mutation in pea xavs

PISUM GENETICS

2011-VOLUME 43

RESEARCH PAPERS

The new xantha-variegata-viredescens mutation in pea

Rozov, S.M.

xavs

Institute of Cytology and Genetics SD RAS Novosibirsk, Russia

Leaf chlorophyll variegation is occasionally observed in many plants. Usually it looks like white, yellow

or dark green spots or irregular regions on leaves. Mutations in both nuclear and organelle genomes

reportedly may cause this leaf variegation (1). It raises a question why and how two neighboring sectors

containing different cell types, a green cell with normal-appearing chloroplasts and a white cell with

abnormal plastids, can be formed in one leaf tissue when the genome of all cells is identical. The precise

mechanism leading to such pseudochimeric chloroplast development in the same leaf tissues remains

poorly understood. One possible explanation for generation of non-identical variegated sectors in each

leaf is a threshold level of factors that arrest proplastid differentiation into chloroplasts (2).

Variegated-type mutants are more rarely observed in plants than the ordinary chlorophyll mutants. In

Arabidopsis thaliana, only four loci are known to cause the variegated phenotype (3). During experimental

mutagenesis, pea mutants with varicgata or

maculata phenotypes appear two orders of

magnitude less frequently than the other

chlorophyll mutants (4, 5). Only four to six

pea variegated mutants have been previously

reported and almost all of them appeared to

be extinct (6). For the above reasons, isolation

of a new pea variegata mutant is of great

interest in the course of studying the

mechanisms of variegated plant appearance

and chloroplast differentiation and survival.
Screening M₂ progeny of the EMS-treated pea

line SGE revealed a new mutant SGE0802

characterized with the strong xantha effect on

shoots and leaflets of the first expanded leaf

Figure 1. SGE0802 (xavs) seedling (A) andproggressive greenish gradient along seedling (B). Digits 1-5 indicate number of node.

(Figure 1).These structures are brilliant

yellow, without any green spots even grown

in the sunlight. Leaflets at the next three nodes (2-4) of

SGE0802 sequentially and progressively become green —

yellow sectors become pale or even white and green

sectors arise in geometric progression from one node to

the next (Figure 2). The 5^t node of the mutant plants

becomes completely green without any allusion of yellow

or other colored sectors. It is notable, that among nearly

5000 plants analyzed, none become fully green at the 4^th or

6^th node — it was always the 5^th node in all cases. The

upper part of the SGE0802 plant is completely normal and

fertility is not affected.

To determine the inheritance type of SGE0802 mutation,

we crossed it with the parental line SGE in both Figure2. The 3^rdnode of a SGE0802 (xavs) seedling. Pale

directions, using the mutant as either the male or female yellow, green and dark green sectors are visible.

PISUM GENETICS

2011-VOLUME 43

RESEARCH PAPERS

parent. In both cases, all F₁ progeny were normal, without any variegation on leaves and all shoots were

green. Segregation results in the F₂ for these crosses are shown in Table 1. In both crosses, the ratio of

normal and variegated seedlings segregated did not differ significantly from 3:1 (p > 0.5). The mutation

appears to be inherited as a single recessive nuclear gene. We designated the observed SGE0802

phenotype as xavs — xantha-variegata-viridescens seedlings.

Table 1. Segregation of F₂ hybrids from crosses of line SGE00802 (xavs) with parental line SGE.


Hybrid	F₂ segregation phenotype		Segregation 3:1	Probability P
Hybrid	*Xavs*	*xavs*	Segregation 3:1	Probability P
SGE0802 x SGE	184	64	0.086	0.7-0.8
SGE x SGE0802	152	56	0.410	0.5-0.6

We tried to localize xavs on the pea genetic map. Line SGE0802 was crossed with a number of tester

lines, but we failed to find linkage of xavs with any of the genetic markers involved. Xavs is not positioned

near genes i, r, d, wb, k, st, b, a, le, gp, wlo, oh, p, pl. The question concerning its location on pea gene map

remains unanswered.

In Arabidopsis thaliana, two genes, var1 and var2 produce the variegation pattern, very similar to the pattern

observed in xavs pea plants, but the Arabidopsis phenotype is constitutive during all plant ontogenesis

(7). Arabidopsis variegated mutations also form three sector types: white, pale green and dark green,

again as was observed in the xavs mutant. In Arabidopsis mutants, dark green sectors contain normal

chloroplasts whereas plastids in the white sectors are vacuolated with obscure inner membrane

structures. Cells of pale green sectors contain plastids of both types. Proplastids in shoot apical

meristems in var Arabidopsis mutants are normal, and abnormal plastids lacking inner membrane

structures begin to form at later stages (3). Var genes belong to a small gene family of 12 nuclear genes

coding for special chloroplast-localized FtsH metalloproteases, homologous to bacterial metalloproteases

and responsible for degradation of photodamaged proteins within the photosynthetic machinery.

Without this protease activity photosynthetic complexes become corrupted and that leads to irreversible

chloroplast degradation (8).

As was shown by scanning electron microscopy, the pea shoot apical meristematic region involves well

differentiated primordium for 4-5 nodes (9). Pea seeds possess a well-developed embryo, so we can

suggest that it also contains well-expanded primordium for the 4-5 first leaves of the future seedling. In

Arabidopsis FtsH genes are stage-specific and only two loci from 12 can produce variegated phenotypes

similar to xavs. We can suggest, that xavs is a homolog of one of the FtsH-family representatives and is

expressed specifically during seed development and maturation, at the stage of initiation of the 1^st lateral

meristem primordium formation. The mutant allele xavs produces a non-functional protease product, but

this product can migrate into proplastids and bind with inner thylakoid membranes where normal

metalloproteases are usually located. A little bit later, during formation of the 4^th and 5^th node meristems,

expression of the other FtsH genes begin and producing two opposite FtsH gradients along primordium

1-5. At primordium 5 the concentration of functional FtsH metalloproteases becomes enough to result in

normal plastid development. The concentration during initiation of the 2^nd - 4^th primordiums is at a

threshold level and that leads to a variegated phenotype appearance where some plastids may or may not

develop normally. certainly, pea gene xavs may produce some factor other than FtsH metalloprotease,

but the general mechanism of variegated phenotype origin must be the same.



PISUM GENETICS	2011-VOLUME 43	RESEARCH PAPERS

References 1. Von Wiren N., Mori S., Marschner H. and Romheld V. 1994. Plant Physiology 106:71-77. 2. Miura E., Kato Y. and Sakamoto W. 2010. Journal of Experimental Botany 61:2433-2445. 3. Sakamoto W., Tamura T., Hanba-Tomita Y., Sodmergen and Murata M. 2002. Genes to Cells 7:769-780. 4. Blixt S., 1972. Agri Hortique Genetica 30:1-293. 5. Lamprecht H. 1974. Monographie der Gattung Pisum. Steiermarkische Landesdruckerei, Graz, Austria. 6. Pisum Genetics Association Genelist: http://www.jic.ac.uk/GERMPLAS/pisum/Zgc4g.htm 7. Martinez-Zapater J.M. 1993. J. Hered. 84:138-140. 8. Sakamoto W. 2003. Genes Genet. Syst. 78:1-9. 9. Gourlay C.W., Hofer J.M.I. and Ellis T.H.N. 2000. The Plant Cell 12:1279-1294.

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