Characteristics and Expression Specificity of RCA Genes in Two Ecotypes of Phragmites australis

doi:10.11983/CBB22204

Abstract

Abstract: (reed) is a cosmopolitan species, with huge biomass and high environmental adaptability. Restricted by its polyploidy genome, it is very difficult to study its unique properties at the genetic level. Using third-generation sequencing and multiple methods, we comprehensively studied the type, structure, expression, and localization patterns of RCA genes (encoding Rubisco activating enzyme) in two reed ecotypes (2n=8x). There are four types of RCA genes in Phragmites genome, all belonging to the RCA2β category. At the transcriptional and protein levels, we adopted immunogold method to detect the localization of RCA proteins. We found that RCAs have obvious ecotype specificity. Compared with swamp reeds (SR), desert-dune reeds (DR) have low RCA expression and RCA2-β2 are preferentially expressed. Six highly expressed RCA isoforms were identified though two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), and were distributed in a high proportion in membrane fraction in DR. This result supports the hypothesis that RCA may transfer to the membrane area as the environment deteriorates, thereby giving RCA a protective function on the chloroplast membrane.

Key words: Phragmites australis, octoploid, desert ecotype, RCA, membrane localization

Qiu Tianhang, Wang An’an, Li Li, Wang Yingchun, Cui Jipeng, Wang Ziyao, Wang Rui, Cui Suxia. Characteristics and Expression Specificity of RCA Genes in Two Ecotypes of Phragmites australis[J]. Chinese Bulletin of Botany, 2023, 58(5): 687-700.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22204

https://www.chinbullbotany.com/EN/Y2023/V58/I5/687

Figures/Tables 8

Figure 1 Classification of predicted RCA genes in Phragmites australis (A) The pairwise genetic distance matrix was generated by using 11 predicted RCA genes of P. australis and 19 known RCA gene sequences of Poaceae (the color key shows genetic distance from close (red) to far (blue); the phylogeny tree in the legend shows the taxonomic relationships of Poaceae subfamilies used in this study; the different subfamilies are highlighted by different colors, while out-group Arabidopsis thaliana shown in black; the data based Poisson clustering tree is on the left, and the phylogeny tree is on the top); (B) Evolutionary analysis of 11 predicted RCA genes of P. australis (all phylogeny trees in this figure were constructed using RCA genomic sequences (including introns and UTR) by MUSCLE alignment of MEGA7 with Neighbor-joining method)

Figure 2 Identification and differential analysis of two RCA genes in Phragmites australis (A) The design of specific primers for the RCA2 genes (the boxes show the primer positions and sequences, the differential nucleotide residues are highlighted in red); (B) The RCA bands from swamp reed (SR) and desert-dune reed (DR) obtained by PCR amplification with genomic DNA as template; (C) Analysis of PaRCA2-β1 and PaRCA2-β2 in sequence and gene structure (the AVID program of mVISTA was used for comparative analysis; the color-coded exon segments were homologous to exons of Arabidopsis thaliana RCA gene)

Figure 3 Expression pattern of RCA in two ecotypes of Phragmites australis (A) Expression level of PaRCA2 genes; (B) Logarithmic histogram (top) and bubble-pie graph (bottom) of transcriptional differences of RCA genes in swamp reed (SR) and desert-dune reed (DR). The proportion of each transcript is annotated with percentage.

Figure 4 Two-dimensional electrophoresis analysis of RCA proteins in two ecotypes of Phragmites australis (A) The protein profiles of the soluble fraction (I) and membrane fraction (II) (six isoforms of PaRCA are marked with white circles and numbers; the white dashed lines indicate the standard molecular weight position of 40 kDa protein); (B) Quantitative analysis of PaRCA, showing 6 RCA isoforms and total RCA content. SR: Swamp reed; DR: Desert-dune reed

Table 1 Localization difference of RCA isoforms in two ecotypes of Phragmites australis

Ecotypes	Soluble fraction (FI) (%)		Membrane fraction (FII) (%)		FII/FI
Ecotypes	L-RCA	S-RCA	L-RCA	S-RCA	L-RCA	S-RCA
Swamp reed (SR)	19.7	54.0	15.9	10.4	0.81	0.19
Desert-dune reed (DR)	11.4	15.9	61.7	11.0	5.41	0.69

Figure 5 Subcellular distribution of RCA immune gold particles in two ecotypes of Phragmites australis SR: Swamp reed; DR: Desert-dune reed; Mt: Mitochondria; V: Vacuoles; Chl: Chloroplast; CW: Cell wall; S: Cytoplasmic stroma; IS: Intercellular stroma; CS: Chloroplast stroma; Gr: Granum. Bars=1 μm

Table 2 The distribution of RCA immune gold particles in the chloroplasts of two Phragmites australis ecotypes (values were means±SD, n=30)

Ecotypes	Membrane fraction		Soluble fraction
Ecotypes	Chl envelope	Thy membrane	Chl stroma	Thy lumen	Others
Swamp reed (SR)	17.5±7.0	29.1±7.5	19.1±6.3	15.7±5.2	18.7±7.3
Desert-dune reed (DR)	26.8±4.2	32.4±5.7	17.1±5.1	11.6±3.3	12.1±4.6
P-value (t-test)	9.95E-08	0.056	0.173	6.85E-04	1.26E-04
DR/SR	1.27		0.76

Figure 6 RCA gene/transcript models of Phragmites australis and Poaceae model plants (A) Gene structure and expression products of AtRCA (the seven exons were shown in different colors, gray represented untranslated region, thin dark gray lines represented introns, and dotted lines represented intergenic sequences); (B) RCA gene model in three Poaceae species (the gene characters were similar between Oropetium thomaeum and Setaria italica; pseudo OsRCA1 had lost its function, while OsRCA2 had two expression products simultaneously); (C) PaRCA gene model. Color labeling and definition in (B) and (C) were aligned with AtRCA by sequence similarity. cTP: Chloroplast transport peptide; N: N-terminal domain; AAA+: ATPase domain; Rub: Rubisco large subunit binding domain; C: C-terminal domain; CTE: Redox related C-terminal extension

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