Exon-intron structure of the 72 sweet orange <i>LEA</i> genes (<i>CsLEAs</i>).

<p>NOI denotes the number of introns, E the exon and I the intron. Numbers on the E and I columns indicate, respectively, the base pair length of the exonic and intronic sequences.</p

Phylogenetic comparison of the complete set of 72 different <i>LEA</i> genes (<i>CsLEAs</i>) encoded in the sweet orange genome.

<p>The different LEA groups are indicated by different colors. Sequence alignment was performed using ClustalX2 and the phylogenetic tree was generated using Bootstrap NJ tree (1,000 resamplings) method and MEGA program (v6.0.5).</p

Chromosomal locations of <i>CsLEAs</i>.

<p>The chromosomal position of each <i>CsLEA</i> was mapped according to the <i>Citrus sinensis</i> Annotation Project (CAP). The <i>CsLEA5/29</i> pair of segmentally duplicated genes is not indicated in the Figure since <i>CsLEA5</i> did not map on any sweet orange chromosome (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145785#pone.0145785.s004" target="_blank">S4 Table</a>). The scale is in Mb. LEA_1 (closed star), LEA_2 (closed triangle), LEA_3 (open star), LEA_4 (open square), LEA_5 (open triangle), DEHYDRIN (open diamond) and SMP (closed diamond).</p

Average number of the <i>cis</i>-elements ABRE (ACGTG), DRE/CRT (G/ACCGAC), MYBS (TAACTG) and LTRE (CCGAC) in promoter region of sweet orange <i>LEA</i> genes from each LEA group.

<p>The <i>cis</i>-elements were analyzed in the 1 kb upstream promoter region of translation start site using the PLACE database.</p

Heatmap of expression of the 72 <i>CsLEA</i> genes in different tissues of sweet orange.

<p>The heatmap was generated using Cluster 3.0 software. The color scale shown represents RPKM-normalized log₂-transformed counts. LEA_1 (closed star), LEA_2 (closed triangle), LEA_3 (open star), LEA_4 (open square), LEA_5 (open triangle), DEHYDRIN (open diamond) and SMP (closed diamond).</p

Conserved motifs in the different groups of sweet orange LEA proteins (CsLEAs).

<p>The conserved motifs were obtained using the MEME program.</p

Expression profiles of the differentially expressed <i>CsLEA</i> genes in response to drought, PEG and salt (NaCl) stresses.

<p>Ratios (log₂) of relative mRNA levels between stressed and control plants in leaves and roots, as measured by qPCR. <i>GAPC2</i> was used as an endogenous control. The bars show means ± SE from three biological replicates.</p

Comparison of the size of the different <i>LEA</i> gene groups in sweet orange (<i>Citrus sinensis</i>), Clementine mandarin (<i>Citrus clementina</i>), <i>Arabidopsis</i> (<i>Arabidopsis thaliana</i>), rice (<i>Oryza sativa</i>), poplar (<i>Populus trichocarpa</i>) and grapevine (<i>Vitis vinifera</i>).

<p>Comparison of the size of the different <i>LEA</i> gene groups in sweet orange (<i>Citrus sinensis</i>), Clementine mandarin (<i>Citrus clementina</i>), <i>Arabidopsis</i> (<i>Arabidopsis thaliana</i>), rice (<i>Oryza sativa</i>), poplar (<i>Populus trichocarpa</i>) and grapevine (<i>Vitis vinifera</i>).</p

Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (<i>Citrus sinensis</i> L. Osb.)

<div><p>The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast (TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic proteins. This study reports a genome-wide survey of <i>MIP</i> encoding genes in sweet orange (<i>Citrus sinensis</i> L. Osb.), the most widely cultivated <i>Citrus</i> spp. A total of 34 different genes encoding <i>C</i>. <i>sinensis</i> MIPs (CsMIPs) were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs) based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of <i>Arabidopsis thaliana</i>. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the <i>CsMIPs</i> possess an exon-intron organization that is highly conserved within each subfamily. <i>CsMIP</i> loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with ‘<i>Candidatus</i> Liberibacter asiaticus’ infection, revealed a tissue-specific and coordinated regulation of the different <i>CsMIP</i> isoforms, consistent with the organization of the stress-responsive <i>cis</i>-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for <i>CsTIPs</i> and <i>CsXIPs</i> during drought stress, and for most <i>CsMIPs</i> during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the <i>CsMIPs</i> functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.</p></div

Expression analysis of the complete set of sweet orange <i>MIPs</i> in response to drought treatment.

<p>Ratios (log₂) of relative mRNA levels between stressed and control plants for all 34 <i>CsMIPs</i> in leaves and roots, as measured by qPCR. <i>GAPC2</i> was used as an endogenous control. The bars show means ± SE from three biological replicates.</p