Series-temporal transcriptome profiling of cotton reveals the response mechanism of phosphatidylinositol signaling system in the early stage of drought stress.

Plants are sessile organisms suffering severe environmental conditions. Drought stress is one of the major environmental issues that affect plant growth and productivity. Although complex regulatory gene networks of plants under drought stress have been analyzed extensively, the response mechanism in the early stage of drought stress is still rarely mentioned. Here, we performed transcriptome analyses on cotton samples treated for a short time (10 min, 30 min, 60 min, 180 min) using 10% PEG, which is used to simulate drought stress. The analysis of differently expressed genes (DEGs) showed that the number of DEGs in roots was obviously more than that in stems and leaves at the four time points and maintained >2000 FDEGs (DEGs appearing for the first time) from 10 min, indicating that root tissues of plants respond to drought stress quickly and continuously strongly. Gene ontology (GO) analysis showed that DEGs in roots were mainly enriched in protein modification and microtubule-based process. DEGs were found significantly enriched in phosphatidylinositol signaling system at 10 min through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, implying the great importance of phosphatidylinositol signal in the early stage of drought stress. What was more, two co-expression modules, which were significantly positively correlated with drought stress, were found by Weighted Gene Co-expression Network Analysis (WGCNA). From one of the co-expression modules, we identified a hub-gene Gohir.A07G058200, which is annotated as "phosphatidylinositol 3- and 4-kinase" in phosphatidylinositol signaling system, and found this gene may interact with auxin-responsive protein. This result suggested that Gohir.A07G058200 may be involved in the crosstalk of phosphatidylinositol signal and auxin signal in the early stage of drought stress. In summary, through transcriptome sequencing, we found that phosphatidylinositol signaling system is an important signal transduction pathway in early stage in response to drought stress, and it may interact with auxin signal transduction through phosphatidylinositol 3- and 4-kinase.

A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress.

DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress.

LeCDJ1, a chloroplast DnaJ protein, facilitates heat tolerance in transgenic tomatoes.

The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1) were investigated using wild-type (WT) and sense transgenic tomatoes. The LeCDJ1 expression was upregulated by 38 °C, 42 °C, 45 °C, NaCl, PEG, methyl viologen (MV) and hydrogen peroxide (H2O2), but not by 30 °C and 35 °C. Meanwhile, LeCDJ1 was involved in the response of plants to abscisic acid (ABA). Under heat stress, the sense plants showed better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), and also less PSII photoinhibition than WT. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher maximum photochemistry efficiency of PSII (Fv/Fm) and D1 protein levels than the SM-untreated WT, suggesting that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, the relatively lower superoxide radical (O2(•-)) and H2O2 levels in the sense plants were considered to be due to the higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, which seemed unlikely dependent on their transcription level. These results indicated that LeCDJ1 overexpression facilitated heat tolerance in transgenic tomatoes.

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation.

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non-photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress-induced photoinhibition and photooxidation, antisense-mediated suppression of zeaxanthin epoxidase transgenic plants and the wild-type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H(2)O(2)), superoxide anion radical (O(2)(•-)) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress-induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.

Expression profiles of a cytoplasmic male sterile line of Gossypium harknessii and its fertility restorer and maintainer lines revealed by RNA-Seq.

The Gossypium harknessii background cytoplasmic male sterility (CMS) system has been used in cotton hybrid breeding in China. However, the mechanism underlying pollen abortion and fertility restoration in CMS remains to be determined. In this study, we used RNA-seq to identify critical genes and pathways associated with CMS in G. harknessii based CMS lines (588A), the near isogenic restorer lines (588R), and maintainer lines (588B). We performed an assembly of 80,811,676 raw reads into 89,939 high-quality unigenes with an average length of 698 bp. Among these, 72.62% unigenes were annotated in public protein databases and were classified into functional clusters. In addition, we investigated the changes in expression of genes between 588A and 588B (588R); the RNA-seq data showed 742 differentially expressed genes (DEGs) between 588A and 588B and 748 DEGs between 588A and 588R. They were mainly down-regulated in 588A and most of them distributed in metabolic and biosynthesis of secondary metabolites pathways. Further analysis revealed 23 pollen development related genes were differentially expressed between 588A and 588B. Numerous genes associated with tapetum development were down-regulated in 588A, implicating tapetum dysplasia may be a key reason for pollen abortion in CMS lines. Also, among DEGs between 588A and 588R, we identified two PPR genes which were highly up-regulated in restorer line. This study may provide assistance for detailed molecular analysis and a better understanding of harknessii based CMS in cotton.

Cloning and Expression Analysis of Eight Upland Cotton Pentatricopeptide Repeat Family Genes.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. Most PPR genes are localized in mitochondria and chloroplasts functioning in regulation of plant growth and development, fertility restoration for cytoplasmic male sterility (CMS), and stress defense. In this study, using in silico cloning and PCR amplification with degenerate primers based on Arabidopsis PPR genes, we cloned eight new full-length PPR genes encoding protein sequences ranging from 458 to 875 amino acids, with 8 to 16 repetitive PPR elements in upland cotton and all of them lack introns. Expression analysis revealed that eight PPR genes were differently expressed in roots, stems, leaves, and floral buds. As for GhI12, its expression in floral buds at days 3-5 was significantly higher in line 777R (restorer line) than in line 777A (CMS line). Further tests with real-time PCR showed that GhI12 expression peaked at day 3 in 777R, followed by a gradual decline, while its expression fluctuated in 777A, peaking at day 5 and day 13. In addition, Gh155c17 and GhI12 were upregulated under salt stress. This is the first report of upland cotton PPR genes involved in salt stress response.

Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco.

Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3-hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was up-regulated under chilling (4 °C), heat (42 °C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O2(·-) levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress.

Overexpression of tomato chloroplast-targeted DnaJ protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco.

DnaJ proteins as co-chaperones have critical functions in biotic and abiotic stress responses, but their biological functions remain largely uninvestigated. This study investigates the function of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ2) using transgenic tobacco. Quantitative real-time polymerase chain reaction analysis showed that LeCDJ2 expression was triggered by salicylic acid (SA), drought and pathogen attack. Ectopic expression of LeCDJ2 in transgenic tobacco reduced the accumulation of superoxide anion radical (O2(-)) and hydrogen peroxide (H2O2) under drought stress. Compared with Vec plants, the maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm), net photosynthetic rate (Pn), and content of D1 protein were relatively higher in transgenic plants. The transgenic plants showes better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrolyte leakage (REL) under drought stress. In addition, overexpression of LeCDJ2 improved the resistance to the pathogen Pseudomonas solanacearum in transgenic tobacco. These results indicate that overexpression of a tomato chloroplast-targeted DnaJ gene enhances tolerance to drought stress and resistance to P. solanacearum in transgenic tobacco.

Heterology expression of the tomato LeLhcb2 gene confers elevated tolerance to chilling stress in transgenic tobacco.

Chilling is one of the most serious environmental stresses that disrupt the metabolic balance of cells and enhance the production of reactive oxygen species (ROS). Light harvesting complex (LHC) proteins had a function in dissipating excess excitation energy and eliminating ROS to maintain the normal physiological function of cells. A tomato (Lycopersicon esculentum) LHC antenna protein gene (LeLhcb2) was isolated. The LeLhcb2-green fluorescent protein (GFP) fusion protein was targeted to the chloroplast of Arabidopsis mesophyll protoplast. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of LeLhcb2 was markedly abundant in leaves and was induced by chilling (4 °C). qRT-PCR analysis and western blot confirmed that the sense gene LeLhcb2 was transferred into tobacco genome and overexpressed. Under chilling stress, the transgenic plants showed not only better growth, higher fresh weight, chlorophyll content, but also lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), compared with the wild type (WT). The maximal photochemical efficiency of PSII (Fv/Fm), non-photochemical quenching (NPQ) and D1 protein content were also higher in the transgenic plants. Furthermore, the relatively lower hydrogen peroxide (H2O2) and superoxide radical (O2(-)) levels in the sense plants were not considered to due to the higher activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD). These results suggested that the overexpression of LeLhcb2 had a key function in alleviating photo-oxidation of PSII and enhanced transgenic tobacco tolerance to chilling stress.

Antisense-mediated depletion of tomato GDP-L-galactose phosphorylase increases susceptibility to chilling stress.

The GDP-L-galactose phosphorylase (GGP), which converts GDP-l-galactose to l-Gal-1-phosphate, is generally considered to be a key enzyme of the major ascorbate biosynthesis pathways in higher plants, but experimental evidence for its role in tomato is lacking. In the present study, the GGP gene was isolated from tomato (Solanum lycopersicum) and transient expression of SlGGP-GFP (green fluorescent protein) fusion protein in onion cells revealed the cytoplasmic and nucleus localization of the protein. Antisense transgenic tomato lines with only 50-75% ascorbate level of the wild type (WT) were obtained. Chilling treatment induced lower increase in AsA levels and redox ratio of ascorbate in antisense transgenic plants compared with WT plants. Under chilling stress, transgenic plants accumulated more malendialdehyde (MDA) and more O(2)(·-), leaked more electrolytes and showed lower maximal photochemical efficiency of PSII (Fv/Fm), net photosynthetic rate (Pn), and oxidizable P700 compared with WT plants. Furthermore, the antisense transgenic plants exhibited significantly higher H(2)O(2) level and lower ascorbate peroxidase (APX) activity. Our results suggested that GGP plays an important role in protecting plants against chilling stress by maintaining ascorbate pool and ascorbate redox state.

Overexpression of a tomato carotenoid ε-hydroxylase gene alleviates sensitivity to chilling stress in transgenic tobacco.

Chilling is one of the most serious environmental stresses that disrupt the metabolic balance of cells and enhance the production of reactive oxygen species (ROS). Lutein plays important roles in dissipating excess excitation energy and eliminating ROS to maintain the normal physiological function of cells. A tomato carotenoid epsilon-ring hydroxylase gene (LeLUT1) was isolated, and the LeLUT1-GFP fusion protein was localized in the chloroplast of Arabidopsis mesophyll protoplast. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of LeLUT1 was the highest in the leaves and was down-regulated by various abiotic stresses in tomato. The transgenic tobacco plants overexpressing LeLUT1 had higher lutein content, which was decreased in cold condition. Under chilling stress, the non-photochemical quenching (NPQ) values were higher in the transgenic plants than in the wild type (WT) plants. Compared with the WT plants, the transgenic plants showed lower levels of hydrogen peroxide (H2O2), superoxide radical (O2(·-)), relative electrical conductivity, and malondialdehyde content (MDA), and relatively higher values of maximal photochemical efficiency of photosystem II (Fv/Fm), oxidizable P700 of PSI, and net photosynthetic rate (Pn). Therefore, the transgenic seedlings were less suppressed in growth and lost less cotyledon chlorophyll than the WT seedlings. These results suggested that the overexpression of LeLUT1 had a key function in alleviating photoinhibition and photooxidation, and decreased the sensitivity of photosynthesis to chilling stress.

Antisense-mediated suppression of tomato thylakoidal ascorbate peroxidase influences anti-oxidant network during chilling stress.

Photosynthesis is a well-established source of reactive oxygen species (ROS) in plants particularly under chilling stress. Ascorbate peroxidase (APXs) plays an important role in the anti-oxidant system by utilizing AsA as specific electron donor to reduce H(2)O(2) to water. In order to investigate the possible mechanisms of ascorbate peroxidsae (APX) in photoprotection under chilling stress, a tomato (Lycopersicon esculentum Mill.) thylakoidal ascorbate peroxidase gene (LetAPX) was isolated and antisense transgenic tomato plants were produced. Under chilling stress, transgenic plants accumulated more H(2)O(2), and showed higher levels of ion leakage and malondialdehyde (MDA), lower net photosynthetic rate (Pn), lower maximal photochemical efficiency of PSII (Fv/Fm) and less content of D1 protein compared with wild type (WT) plants. On the other hand, after chilling stress, transgenic plants showed higher reduced ascorbate (AsA) and activities of catalase (CAT) and superoxide dismutase (SOD) than those in WT plants, and the expression of several known stress-responsive and antioxidative genes was also higher at the end of chilling treatment. These results suggested that the suppression of LetAPX gene induced compensatory anti-oxidant mechanisms in tomato, and inactivation of tAPX may have a regulatory role in facilitating redox signaling pathways under chilling stress. Furthermore, transient increases in ROS levels also have a vital role in stress signaling and thereby in the survival of plants under chilling conditions.

Antisense-mediated depletion of tomato chloroplast glutathione reductase enhances susceptibility to chilling stress.

A tomato (Lycopersicon esculentum Mill.) chloroplast glutathione reductase gene (LeGR) was isolated and antisense transgenic tomato lines were obtained. Under chilling stress, transgenic plants accumulated more H(2)O(2), leaked more electrolyte and showed lower net photosynthetic rate (Pn), maximal photochemical efficiency of PSII (Fv/Fm) and oxidizable P700 compared with wild-type (WT) plants. Transgenic seedlings were more suppressed in fresh-weight growth and lost more cotyledon chlorophyll. The decrease in the activity of ascorbate peroxidase (APX) was implied to be potentially relevant to the greater accumulation of H(2)O(2) in transgenic plants. Chilling treatment induced more decrease in the level of reducted glutathione (GSH) and redox ratio of glutathione in transgenic plants than in WT plants, but aroused more increase in GSSG in transgenic plants than in WT plants. Total glutathione displayed no change. Besides, chilling stress resulted in greater decreases in the level of reducted ascorbate (AsA), total ascorbate and redox ratio of ascorbate in transgenic plants than in WT plants, but led to equivalent degree of dehydroascorbate (DHA) increase in WT and transgenic plants. These assessments of glutathione-ascorbate cycle revealed that the decrease of glutathione reductase activity in transgenic plants affected glutathione regeneration, and consequently affected ascorbate regeneration and total ascorbate content. This resulted in a greater accumulation of H(2)O(2) and an enhanced sensitivity to chilling stress in transgenic plants. Moreover, a putative concept model of ecophysiological reaction was discussed.