Prunus Knotted-like Genes: Genome-Wide Analysis, Transcriptional Response to Cytokinin in Micropropagation, and Rootstock Transformation

Abstract

Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6-0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression.

Keywords: 6-benzyladenine; KNOX; Prunus spp.; bioinformatics; gene expression; genetic transformation; in vitro shoot multiplication; rootstocks.

Figure 1

(A) Phylogenetic tree of PRUNOX deduced proteins, clustered into classes I, II, and M (blue, orange, and green shades) and named based on the presence of A. thaliana (bold) members in the clade. (B) Plots of amino acid variability within KNOX subgroups. The peach proteins were schemed (top of each panel) together with key domains (colored boxes) and used as reference for KNOX of other species. Identity variation (percent) of amino acids within Prunus spp. proteins (Y-axis) refers to amino acid positions (X-axis). Orange, KNOX1 (PF03790); red, KNOX2 (PF03791); grey, ELK (PF03789); blue, homeodomain (PF05920).

Figure 2

Comparative schematic maps of KNOX genome distribution occurring in species of the Armeniaca section (e.g., P. mume, Pm) vs. that in other Prunus spp. (e.g., P. persica, Pp). The gene KNOPE2.1 is located in the unplaced scaffold1397 (Scf1397) in P. mume but in Chr 7 in P. armeniaca. The scale on the left represents chromosome lengths in megabases (Mb).

Figure 3

Exon–intron structure and conserved motifs in PRUNOX genes. The organization of each class was compared to A. thaliana counterparts (grey-shaded). Boxes, coding sequences; gray lines, introns. KNOX conserved motifs are colored.

Figure 4

Cytokinin binding motifs residing on PRUNOX genes’ promoters of P. persica and P. dulcis (left and right panels). The score accounts (covering 1.5 kb region before the start codon in Table S4) refer to the experimentally determined extended (ECRM; blue) and core (CRM; light blue) motifs [38], as well as octameric sequences (yellow/orange/brown) enriched in CK-responsive promoters [39].

Figure 5

Prunus genome-wide transcription analysis of PRUNOX genes active in aerial organs. The expression profiles refer to 10 species through heat maps of the z-scores, where orange and blue indicate higher and lower expression, respectively. fl., flower.

Figure 6

Effects of BA supply on GF677 microcuttings (A,F,K). Explants of the GF677 rootstock were grown on media without BA (A–E) or containing BA at concentrations of 1.7 (F–J) and 5.1 µM (K–O). Ten days post-treatment, the effects of BA dosage in increasing the numbers of side shoots and leaves (compare (F,K) vs. (A)) were visible. (B–E,G–J,L–O) Histological analyses of longitudinal sections at early stages (3 days after treatment). Median section through the dome of control (B) and BA-treated apices (G,L). Leaf axillary buds showed a plethora of phenotypes, including normal (C,D,H,M), elongating (I), and swollen (O). Section of stems portions adjacent to medium (E,J). Bar sizes: 0.5 cm in (A,F,K); 150 µm in (D,I,L–N); 160 µm in (B,G,H,J,L,O); 170 µm in (C).

Figure 7

Gene expression analyses of KNOPE and marker genes under different BA dosages. Expression levels of stress- (A,B) and CK-responsive (C,D) markers and of KNOPE (E,H) and BELL (I,J) genes in response to 1.7 µM (A,C,E,G,I) and 5.1 µM (B,D,F,H,J) of BA at 24 and 72 h post-treatment (hpt). Each value represents the mean ± standard error of three replicates. For each gene, different letters mean significant differences (p ≤ 0.05).

Figure 8

Correlation plot between TALE and marker gene expression levels. Correlogram representing Pearson’s correlation coefficients (r) between TALE (PRUNOXI: STMlike1, STMlike2, KNOPE1, KNOPE2, KNOPE2.1, KNOPE6; PRUNOXII: KNOPE3, KNOPE4, KNOPE7; BELL: PpBEL1, PpBLH1, PpBLH2, PpBLH3, PpBLH5, PpBLH6, PpBLH8) and marker (stress-responsive: PpCAT1, PpCAT2; CK-responsive: PpCKX6, PpARR12, PpHK1) expression levels. Heat map is used to indicate the strength of correlation between the variables with ordering determined by hierarchical clustering. Red and blue indicate negative and positive correlations, respectively. Only significant (p ≤ 0.05) Pearson’s coefficients were reported in the colored squares. *, **, and *** = significant at p ≤ 0.05, 0.01, and 0.001, respectively.

Figure 9

Genetic transformation of Gisela 6. (A–J) Examples of GFP fluorescence analysis in leaves and roots of Gisela 6 transgenic clones. (A–D) P1 clone. (E–H) P3 clone. Analysis of leaf margins (A,C,E,G) and lateral roots (B,D,F,H) under visible microscopy (A,B,E,F) and fluorescence (C,D,G,H). (K–Q) 35S:KNOPE1 phenotypes in Gisela 6. Regenerated clones (K,N) and details of leaf margins (L–O) and lamina vasculature (M,P) in nontransformed (K–M) and 35S:KNOPE1 (N–P) lines. (Q) 35S:KNOPE1 clone that reverted phenotype with time. (R) Vector schemes of pCAMBIA1302 (above) and pBA002 + KNOPE1 (below); dark grey bars, probes used in Southern blots which are reported in Figure S3; arrowheads, primers used to check for transgene integrity (black) and expression (red). (S) Upper panel, a check for p35S:KNOPE1:NOSt cassette integrity by PCR with gDNA. Six clones were rescued and analyzed; on the left, size of bands of DNA ladder in base pairs (bp); on the right, the amplicon size is specified. Mid panel, a check for KNOPE1 transgene expression in the six clones by RT-PCR using leaf blade RNA. Peach (Chiripa) KNOPE1-specific primers fell between the 5’UTR and the first exon (R). The constitutive RPII expression was assayed to check for correct retrotranscription and usage of equal cDNA amounts. The amplicon sizes are reported.