Variability of Chloroplast DNA of Extranuclear Sunflower Mutants

Corresponding Author: Lyubov Getmantseva Department of Biotechnology, Don State Agrarian University, Persianovskiy, Russia Email: ilonaluba@mail.ru Abstract: A comparative analysis of full-genome sequences of chloroplast DNA (cpDNA) of the original inbred line 3629 and three extranuclear mutants, which were obtained by the method of mutagenesis induced with N-nitroso-N-methylurea (NMU) and characterized by different level of chlorophyll insufficiency (en:chlorina-7-yellow-green leaves; chlorophyll content (a + b)-67.8% with respect to the line 3629, variegated-10-leaves with white zones; chlorophyll content (a + b)-2.9% with respect to the line 3629 and variegated-13-leaves with yellow zones; chlorophyll content (a + b)-6.1% with respect to the line 3629), has been carried out. Single-parent maternal inheritance of chlorophyll defects was confirmed by analysis of progeny obtained from reciprocal crossbreedings between the original line 3629 and mutants. Chlorophyll mutants carried modified cpDNA unique for each mutant. We anticipate that chlorophyll defect of en:chlorina-7 may control the observed non-synonymous mutations (transitions) in the genes rpoB, psaA and psbB, which encode β-subunit of RNA-polymerase, the A1 apoprotein of chlorophyll a of the photosystem I, P700 and 47 kDa protein of the photosystem II respectively. In variegated-10, it may control mutations in the genes rpoA and rpoC2, which encode α and β” subunits of RNA-polymerase and in variegated-13-two mutations in the ycf3 gene that encodes photosystem I assembly factor.


Introduction
Presently, a variety of DNA markers is used to assess plant gene polymorphisms (Wong et al., 2009;El-Awady et al., 2012;Usatov et al., 2014;Bhavsar et al., 2015). They are also effectively applicable in order to mark individual traits of plants (Poczai et al., 2013). However, analysis of full-genome sequences provides more accurate results.
It is common knowledge that mutants may be used as a suitable model for studying the "gene-trait" problem and extranuclear mutants are not excluded. However, their importance becomes even higher as biogenesis, functions of chloroplasts and mitochondria and their photosynthetic and respiratory activity are subjected to double nuclear-organelle regulation. Therefore, genetic analysis of these inheritable modifications allows us to reveal not only cytogene determined structural components of organelle, but also principles of nuclear-cytoplasmic relationships (Strand et al., 2003;Barajas-Lopez et al., 2013).
In the Southern Federal University, we obtained a series of chlorophyll mutations by mutagenesis of seeds of the inbred line 3629 induced with N-nitroso-N-methylurea (NMU) (Beletskii et al., 1969). The selected survivable mutants were referred to two phenotypic classes: Mutants with yellow-green leaves (chlorina) and poecilophyllous chimera with yellow and white zones on their leaves. Extranuclear nature of these mutations was confirmed by hybrid analysis. Extranuclear chlorophyll mutants are considered to be the appropriate model to study the role of chloroplast genome in chlorophyll biosynthesis and biogenesis of photosynthetic apparatus (Vezitskii et al., 1999;Rassadina et al., 2001;Usatov et al., 2001;2004;Rassadina et al., 2005).
Previously, it was attempted to map the mutations using the restriction analysis of cpDNA of the line 3629 and en:chlorina mutants (Triboush et al., 1999). In particular, it was shown that cpDNA of the en:chlorina-7 mutant contained an additional HindIII endonuclease restriction site as compared with the line 3629. However, this method appeared not to be sufficiently effective to locate the mutations. It became clear that more distinctive approaches are needed to locate mutations in cpDNA (Henry et al., 2014).
Threfore, the goal of our work was to study cpDNA variability in extranuclear chloroplast sunflower mutants with different level of chlorophyll defects, using the fullgenome sequencing.

Рlants
The objects of our study were plants of the original inbred line 3629 and three extranuclear mutant lines characterized by different level of chlorophyll insufficiency (en:chlorina-7-yellow-green leaves, variegated-10-leaves with white zones, variegated-13-leaves with yellow zones), which were obtained by mutagenesis induced with N-nitroso-N-methylurea (NMU).

Analysis of Hybrids
Analysis of hybrids was performed under the field conditions. The maternal plants were pollinated after sterilization and then, the plants were isolated. Plants were grown under the conditions of selection farm, in the 10 m plots of land with the area 40×60 cm.
The amounts of chlorophyll and carotinoids were assessed by the absorption spectra of 85% acetone extracts of leaves collected at the budding stage (Shlyk, 1971).
To obtain the full-genome sequences of cpDNA total DNA was isolated from green leaves of the original line 3629, yellow-green leaves of the en:chlorina-7 mutant and from white and yellow zones of leaves of the variegated-10 and variegated-13 mutants respectively. Total DNA was isolated by the modified CTAB method (Doyle and Doyle, 1990).
Polyvinylpyrrolidone 40000 and sodium metabisulfite were added to the extraction buffer; for removing RNA, the DNA fractions obtained were incubated for 1 h at 37°C in the presence of RNase A.

Sequencing
Full-genome sequencing of cpDNA was performed in the HiSeq 2000 sequenator ("Illumina", USA) with the length of reading 100+100. The obtained sequences were mapped onto the chloroplast genome of sunflower, line НА383 [GenBank NC_007977 (Timme et al., 2007)]. The results were analyzed with the program CLC Genomics Workbenchv. 6.0.4. The obtained sequences were aligned with the program BioEdit 7.0.9.0. Synonymous and non-synonymous mutations were identified with the program ExPASy (http://web.expasy.org/translate/).
To perform the direct sequencing, the DNA amplicons were obtained and purified (Werle et al., 1994). Sequencing of amplicons was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) and ABI Prism 3130xl Genetic Analyser (Applied Biosystems, USA). In each line 11-15 plants were studied.

Results
The results of reciprocal crossbreedings of the chlorophyll mutants variegated-10, variegated-13 and en:chlorina-7 with plants of the original line 3629 are shown in Table 1 and 2. If mutant poecilophyllous plants were pollinated with pollen of green plants from the line 3629, their progeny was split in F1 into three types of seedlings: Green and variegated white and yellow (Table 1). If en:chlorina-7 mutant plants were pollinated, only mutant phenotype was inherited (Table  2). In further generations the self-pollinated poecilophyllous mutants split with nearly the same ration as in F1, while the chlorina phenotype remained unchanged. The reciprocal crossbreeding, i.e., when green plants of the line 3629 were used as maternal line and the mutant form was the paternal one, produced totally green progeny that did not split in further generations. Progeny of green plants produced by poecilophyllous mutants also remained unchanged. Table 1. Reciprocal crossbreedings of the variegated -10 and variegated -13 mutants with green plants of the original line 3629 Green Chlorina  Green  Chlorina  en:chlorina-7×3629  0  44  0  412  3629×en:chlorina-7  41  0  786  0   Table 3. The level of chlorophylls (a + b) and carotinoids in the leaf tissue of sunflower mutants Chlorophyll (a+b) Tissue phenotype mg/g of dry weight % from the control mg/g of dry weight % from the control 3629 Green 8 We performed a comparative analysis of chlorophyll and carotinoid level, because mutant plants were phenotypically different, especially by leaf color that differed from white to yellow green, from the original green plants (Table 3). It was shown that yellow-green leaves of the en:chlorina-7 mutant contained 5.78 mg g −1 of the dry weight of chlorophylls a and b. Total concentration of green pigments in the white leaf tissue of the variegated-10 mutant was 0.25 mg g −1 of dry weight. Yellow tissue of the variegated-3 mutant contained 0.52 mg chlorophyll per 1 g of dry weight. The total level of chlorophylls in the original green plants of the line 3629 was 8.53 mg g −1 of dry weight. The level of carotinoids in mutant plants was also lower than in the original green plants (Table 3).
The analysis of cpDNA structure of the line 3629 and the en:chlorina-7 revealed the following differences: One deletion (TA) in the intergene region (petA-psbJ) and seven single nucleotide polymorphisms, three of which were located in the genes rpoA, rps11 и ycf1 (synonymous), one in the intergene region rps12-clpP (synonymous) and the other in the genes rpoB, psaA, psbB (non-synonymous). The latter were represented by the transition (C/T) in the gene, encoding β-subunit of RNA-polymerase (Ser138Leu), in which the reactive center located and G/A substitutions in the gene, encoding the A1 apoprotein of chlorophyll a (Thr528Ile), which is involved into the photosystem I and C/T substitution in the gene, encoding the 47 kDa protein (His157Tyr) involved into the photosystem II (Table 4).
Structure of cpDNA of the variegated-10 mutant also differed from that of the line 3629 by two microsatellite loci, (C) 11 and (T) 15 and by seven SNPs. Two of the SNPs were represented by non-synonymous substitutions: G for A (rpoA (Thr203Ile)) and C for T (rpoC2 (Leu768Phe)), which were located in the genes, encoding α and β" subunits of RNA-polymerase respectively (Table 4). These subunits represent a functionally important part of chloroplast RNA-polymerase, which participates in cpDNA transcription (Kremnev and Strand, 2014;Börner et al., 2015;Pfannschmidt et al., 2015). The other five SNPs, apparently, do not lead to modifications in the translation products, being either synonymous substitutions in the psaA and ndhG genes or located in the intergene regions of chloroplast genome (ycf6-psbM, rpl16-rps3 and rpl32-ndhF).
We identified two polymorphous microsatellite loci, (C) 11 и (T) 15 , in cpDNA of the variegated-13, which were similar to those of the variegated-10 mutant and three unique SNPs. One of them is located in the noncoding region of chloroplast genome (intergene region trnL-UAG-rpl32), while two non-synonymous SNPs-Ala91Thr and Glu65Lys are located in the ycf3, which encodes the photosystem I assembly factor. Ycf3 is known to work as a chaperone, which specifically interacts with PsaA and PsaD subunits during assembly of the photosystem I complex (Naver et al., 2001;Landau et al., 2009).

Discussion
Former electron microscopic analysis of cell and organelle of mutant leaves revealed modifications of chloroplast ultrastructure that led to chlorophyll defects and, subsequently, to the decrease in photosynthetic activity (Usatov et al., 2004;Rassadina et al., 2005). It was shown that poecilophyllous mutants lacked thylakoid system and thus, suffered chlorophyll insufficiency. They often contained osmiophilic granule represented by non-structural aggregates of proteinlipid components of intracellular membranes. Lack of pigments well correlated with breaking of plastid structure of survivable mutants en:chlorina: Some of lamellae were vacuolated, general disorganization of the lamella structure was observed and thylakoid granae were poorly developed as compared with chloroplast of the line 3629.
It was also shown that the first traits of chlorophyll deficiency of the variegated-10 mutant are synchronic decrease in the synthesis of chlorophyll precursor, 5aminolevulinic acid, lower ratio of a and b chlorophylls in comparison with the line 3629 and reduction in the photosystem II chlorophyll fluorescence (Usatov et al., 2004). The observed defects increased as mutants developed and eventually led to gradual destruction of the photosystem II and light-consuming complexes. The trait of chlorophyll deficiency of the variegated-10 was unstable and could either occur or disappear, depending on the plant growing conditions, such as temperature and light regimes. However, the dependence of the chlorophyll deficiency trait on light and temperature conditions was observed at early stages of pigment containing tissue development only. At this stage, low illumination not only did not induce chlorophyll insufficiency, but rather prevented destructive processes, when the mutant was transferred to the conditions, which induced most sever pigment abnormality. Such manifestation of the mutant trait may be due to defects of plastogene expression that control synthesis of pigment and protein components of the photosynthetic apparatus, nevertheless our study showed that mutations were located in structural genes of cpDNA.

Conclusion
A comparative analysis of the full-size sequences of cpDNA of the original inbred line 3629 and three extranuclear NMU-induced mutants characterized by different level of chlorophyll insufficiency (en:chlorina-7-yellow-green leaves, variegated-10-leaves with white zones and variegated-13-leaves with yellow zones) has been carried out. Each of the chlorophyll mutants carried uniquely modified cpDNA. We suggest that chlorophyll defect of en:chlorina-7 may control non-synonymous transitions in the rpoB, psaA, psbB genes, which encode β-subunit of RNA-polymerase, A1 apoprotein of chlorophyll a of the photosystem I P700 and the 47 kDa protein of the photosystem II respectively. In the variegated-10 mutant it may control mutations in the rpoA and rpoC2 genes, which encode α and β" subunits of RNA-polymerase and in variegated-13, -two mutations in the ycf3 gene, which encodes the photosystem I assembly factor.

Ethics
This article is original and contains unpublished materials. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.