Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.Embrapa (Macroprograma)Embrapa (Macroprograma)CNPq (Brasilia, Brazil)CNPq (Brasilia, Brazil)FAPESP (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)CAPES (Brasilia, Brazil)CAPES (Brasilia, Brazil)FAPESB (Salvador, Brazil)FAPESB (Salvador, Brazil)CAPES PNPDCAPES/PNP

Genome-wide characterization and expression analysis of citrus NUCLEAR FACTOR-Y (NF-Y) transcription factors identified a novel <i>NF-YA</i> gene involved in drought-stress response and tolerance

<div><p>Nuclear factor Y (NF-Y) is a ubiquitous transcription factor found in eukaryotes. It is composed of three distinct subunits called NF-YA, NF-YB and NF-YC. NF-Ys have been identified as key regulators of multiple pathways in the control of development and tolerance to biotic and abiotic factors. The present study aimed to identify and characterize the complete repertoire of genes coding for NF-Y in citrus, as well as to perform the functional characterization of one of its members, namely <i>CsNFYA5</i>, in transgenic tobacco plants. A total of 22 genes coding for NF-Y were identified in the genomes of sweet orange (<i>Citrus sinensis</i>) and Clementine mandarin (<i>C</i>. <i>clementina</i>), including six CsNF-YAs, 11 CsNF-YBs and five CsNF-YCs. Phylogenetic analyses showed that there is a NF-Y orthologous in the Clementine genome for each sweet orange NF-Y gene; this was not observed when compared to <i>Arabidopsis thaliana</i>. CsNF-Y proteins shared the same conserved domains with their orthologous proteins in other organisms, including mouse. Analysis of gene expression by RNA-seq and EST data demonstrated that <i>CsNF-Y</i>s have a tissue-specific and stress inducible expression profile. qRT-PCR analysis revealed that <i>CsNF-YA5</i> exhibits differential expression in response to water deficit in leaves and roots of citrus plants. Overexpression of <i>CsNF-YA5</i> in transgenic tobacco plants contributed to the reduction of H₂O₂ production under dehydration conditions and increased plant growth and photosynthetic rate under normal conditions and drought stress. These biochemical and physiological responses to drought stress promoted by <i>CsNF-YA5</i> may confer a productivity advantage in environments with frequent short-term soil water deficit.</p></div

Heatmap of expression of <i>CsNF-Y</i>s in different tissues of sweet orange.

<p>The color scale shown represents RPKM-normalized log₂-transformed counts.</p

Citrus <i>NF-Y</i> transcription factor encoded genes.

<p>Citrus <i>NF-Y</i> transcription factor encoded genes.</p

Physiological analysis of <i>CsNFYA5</i>-overexpressing transgenic lines.

<p>Control (WT and PC) and <i>CsNFYA5</i> transgenic (NF12, NF15, NF16, NF20 and NF22) lines were subjected to control (irrigated) and drought treatments under greenhouse conditions. The data are means ± SE of five plant replicates per treatment for each line. Statistically significant differences at <i>P</i> ≤ 0.01 (**) or <i>P</i> ≤ 0.05 (*) between control (WT) and <i>CsNFYA5</i> transgenic lines, at the respective water treatment, are indicated.</p

Multiple alignments and predicted structure of conserved regions of CsNF-Y family members.

<p>Sequence alignments among the highly conserved domains of NF-YA (<b>A</b>), NF-YB (<b>B</b>) and NF-YC (<b>C</b>) proteins of <i>C</i>. <i>sinensis</i> (Cs) and <i>Mus musculus</i> (Mouse). The DNA binding domain and the domains required for interaction with the other subunits previously defined in yeast and mammals are indicated. The secondary structures, alpha-helices (rectangles) and coils (black lines), are represented on the bottom of the alignment, based on Romier et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199187#pone.0199187.ref055" target="_blank">55</a>]. Predicted structures of NF-Y conserved regions using the protein homology modeling of Phyr2 <b>(D)</b>. Images of the models were coloured in a rainbow from blue to red from the N-terminus to the C-terminus.</p

Expression analysis of <i>CsNF-Ys</i>.

<p>Heatmap of expression of <i>CsNF-Ys</i> in response to different stress treatments <b>(A)</b>. The color scale shown represents TPM-normalized log₂-transformed counts. qRT-PCR expression analysis of <i>CsNF-YA5</i> <b>(B)</b>. <i>CsNF-YA5</i> mRNA abundance in leaves and roots of sweet orange plants grafted on Rangpur lime, subjected to control (irrigated) and drought treatments. The data are means ± SE of three biological replicates in which <i>β-actin</i> transcripts were used as internal controls.**Significantly different from control treatment at <i>P</i> ≤ 0.01.</p

<i>In vitro</i> drought stress tolerance assay of <i>CsNFYA5</i>-overexpressing transgenic lines.

<p>Representative phenotypes of control (WT and PC) and <i>CsNFYA5</i> transgenic (NF12, NF15, NF16, NF20 and NF22) lines grown under control and PEG treatments for 30 days <b>(A, B)</b>. Shown from left to right in panel B: WT, PC, NF12, NF15, NF16, NF20 and NF22. Seedling biomass and root length, respectively, of control and transgenic lines under control and PEG treatments for 30 days <b>(C, D)</b>. The data are means ± SE of five technical replicates composed of fifteen seedlings for each line. Total thiobarbituric acid reactive substances (TBARS) concentration in seedlings of WT and transgenic line NF16 under control and PEG treatments for 30 days <b>(E)</b>. The data are means ± SE of six technical replicates composed of fifteen seedlings for each line. Statistically significant differences at <i>P</i> ≤ 0.01 (**) or <i>P</i> ≤ 0.05 (*) between control (WT) and <i>CsNFYA5</i> transgenic lines, at the respective treatment, are indicated.</p