Kunzhi Li

Differential Gene Expression Analysis of Yunnan Red Pear, Pyrus Pyrifolia, During Fruit Skin Coloration

The color of fruit skin is an important quality parameter, and in many plants, it is the result of coordinative regulation of the anthocyanin pathway. To characterize the mechanism involved in fruit peel coloration of Yunnan red pear (Pyrus pyrifolia), we constructed a subtractive cDNA library using the suppression subtractive hybridization (SSH) technology. cDNA of red peel exposed to sunlight (for 2, 4, 6, and 8xa0days) was subtracted from that of white skin unexposed to sunlight. Over 100 differentially expressed ESTs were obtained, putatively involved in primary and secondary metabolism, stress, and defense response. Expression analysis using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) for 13 genes was performed with two pear cultivars, light-skinned ‘Zaobaimi’ and red-skinned ‘Yunhong-1’, which had been bagged and then exposed to sunlight for 0, 1, 2, 3, 5, and 7xa0days before harvest. This analysis showed that genes encoding for a metallothionein-like protein and a NADP-malic acid enzyme were constitutively expressed, whereas other selected genes were either down- or up-regulated. Semiquantitative RT-PCR analysis for 7 anthocyanin biosynthetic pathway genes and 3 putative regulatory genes was also performed. Results showed that an R2R3 MYB transcription factor PyMYB10 was up-regulated in both the less-colored pear ‘Zaobaimi’ and well-colored red pear Yunhong-1 after the bag was removed, but that kinetics differed between cultivars. Other anthocyanin-related genes appeared to be coordinately regulated by the MYB–bHLH–WD40 complex. DFR and ANS genes seemed to be limiting factors for the peel coloration of less-colored pear ‘Zaobaimi’, while all biosynthetic steps are up-regulated by 7xa0days after bag removal in red fruit. This study suggests the regulation of red pear coloring is via differential effects of the MYB–bHLH–WD40 complex on the pear anthocyanin pathway genes.

Overexpression of malate dehydrogenase in transgenic tobacco leaves: enhanced malate synthesis and augmented Al-resistance.

Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in the present study. Our data indicated that an increase (120–130%) in MDH-specific activity in leaves led to an increase in malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared with the WT plants under the acidic (pH 4.5) conditions irrespective of 300xa0μM Al3+ stress absence or presence. After being exposed to 25xa0μM Al3+ in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance.

Transcriptional and physiological changes of alfalfa in response to aluminium stress

Medicago sativa is an excellent pasture legume, but it is very sensitive to aluminium (Al) toxicity. To better understand the mechanism of M. sativa sensitivity to Al, a forward suppression subtractive hybridization (SSH) cDNA library for an Al-sensitive cultivar, M. sativa L. cv. Yumu No. 1 (YM1), under 5 μ m Al stress over a 24 h period was constructed to analyse changes in its gene expression in response to Al stress. Sequence analysis for the SSH cDNA library generated 291 high-quantity expression sequence tags (ESTs). Of these, 229 were known as functional ESTs, 137 of which have already been reported as Al response genes, whereas the other 92 were potentially novel Al-associated genes. The up-regulation of known Al resistance-associated genes encoding the transcription factor sensitive to proton rhizotoxicity 1 ( STOP1 ) and malate transporter MsALMT1 (Al-activated malate transporter) as well as genes for antioxidant enzymes was observed. Reverse transcription polymerase chain reaction analysis validated the reliability of the SSH data and confirmed the up-regulated expression of STOP1 and MsALMT1 under 5 μ m Al stress. The analysis of physiological changes indicated that hydrogen peroxide (H 2 O 2 ) and malondialdehyde levels were elevated rapidly under 5 μ m Al stress, suggesting that severe oxidative stress occurred in the YM1 roots. The up-regulation of antioxidant-related genes might be an important protective mechanism for YM1 in response to the oxidative stress induced by 5 μ m Al toxicity. Al-induced malate exudation was increased drastically during the early period after Al treatment, which might have been due to the up-regulation and function of MsALMT and STOP1 . However, malate exudation from the YM1 roots declined quickly during the subsequent period, and a gradual decrease in malate content was simultaneously observed in the YM1 roots. This result is in agreement with the observation that organic acid metabolism-associated enzymes such as phosphoenolpyruvate carboxylase, citrate synthase and malate dehydrogenase were not present in the SSH library. This might be a major reason for the YM1 sensitivity to Al.

Identification of Early Nitrate Stress Response Genes in Spinach Roots by Suppression Subtractive Hybridization

To better understand the molecular basis of plant responses to nitrate stress, suppression subtractive hybridization (SSH) was used to identify the potential important or novel genes involved in the early stage of spinach responses to nitrate stress. Complementary DNAs (cDNAs) of 15xa0min, 1xa0h, 2xa0h, 6xa0h and 24xa0h of 160xa0mM nitrate stress treatment seedlings were used as tester, and cDNAs of normal nutrient solution seedlings of the same time were used as driver. The SSH analysis identified 189 non-redundant putative nitrate stress responsive cDNAs out of 798 clones. These ESTs were categorized into 12 functional groups, with the largest group of genes involved in metabolism, followed by the group of genes related to cell rescue, defense and virulence. Reverse transcriptase (RT)-PCR was conducted for several genes, confirming the induction by nitrate stress. The results indicated that osmolyte biosynthesis genes, reactive oxygen scavengers, transporters, signaling components and transcription factor played important roles in fighting against nitrate stress. The diversity of the putative functions of these genes indicated that nitrate stress resulted in a complex response in spinach plants.

Overexpression of spinach non-symbiotic hemoglobin in Arabidopsis resulted in decreased NO content and lowered nitrate and other abiotic stresses tolerance.

A class 1 non-symbiotic hemoglobin family gene, SoHb, was isolated from spinach. qRT-PCR showed that SoHb was induced by excess nitrate, polyethylene glycol, NaCl, H2O2, and salicylic acid. Besides, SoHb was strongly induced by application of nitric oxide (NO) donor, while was suppressed by NO scavenger, nitrate reductase inhibitor, and nitric oxide synthase inhibitor. Overexpression of SoHb in Arabidopsis resulted in decreased NO level and sensitivity to nitrate stress, as shown by reduced root length, fresh weight, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm), and higher malondialdehyde contents. The activities and gene transcription of superoxide dioxidase, and catalase decreased under nitrate stress. Expression levels of RD22, RD29A, DREB2A, and P5CS1 decreased after nitrate treatment in SoHb-overexpressing plants, while increased in the WT plants. Moreover, SoHb-overexpressing plants showed decreased tolerance to NaCl and osmotic stress. In addition, the SoHb-overexpression lines showed earlier flower by regulating the expression of SOC, GI and FLC genes. Our results indicated that the decreasing NO content in Arabidopsis by overexpressing SoHb might be responsible for lowered tolerance to nitrate and other abiotic stresses.

Molecular cloning and characterisation of a germin-like protein gene in spinach (SoGLP)

Summary Germin-like proteins (GLPs) are ubiquitous plant glycoproteins that have been implicated in various plant physiological and developmental processes. In this study, a cDNA clone, designated SoGLP, encoding a germin-like protein from spinach (Spinacia oleracia L.) was isolated and characterised. SoGLP encodes a 208-amino-acid polypeptide with a predicted molecular mass of 22.54 kDa and a pI of 5.95. Phylogenetic analysis indicated that SoGLP belonged to sub-family 3 of the GLP family. Sub-cellular localisation of an SoGLP-GFP fusion protein appeared to detect the protein on the cell walls in transgenic tobacco plants. Real-time quantitative RT-PCR analysis showed that the level of expression of SoGLP in the salt-resistant spinach cultivar (‘Chaoji’) was generally higher than in the salt-sensitive spinach cultivar (‘Daye’) grown in 160 mM nitrate ions for 0.5, 3.0, or 6.0 h. Expression of the SoGLP gene was also induced by other abiotic stresses including polyethylene glycol (PEG), NaCl, salicylic acid (SA), or H2O2 treatment. Our results indicate that SoGLP could play important roles during high nitrate stress or under other abiotic stresses.

Effects of exogenous H2S on the germination of tomato seeds under nitrate stress

Summary In this study, the effects of the exogenous H2S donor, NaHS, on the germination of tomato seeds under nitrate (NO3 -) ion stress were investigated. The results showed that under increased nitrate ion stress (range 0 - 200 mM), the germination percentage of tomato seeds decreased from 92.3% to 1.0%. The application of NaHS, up to 100 ?M, alleviated the inhibitory effect of nitrate stress by enhancing seed germination up to 1.3-fold compared to the nitrate stress treatment alone. Further study showed that malondialdehyde concentrations and the accumulation of reactive oxygen species declined significantly after all NaHS treatments. Amylase, superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase activities increased significantly after 100 ?M NaHS treatment. In addition, nitric oxide (NO) fluorescence increased when NaHS was added to the nitrate solution. Our results suggest that exogenous H2S can alleviate the damage caused by nitrate stress, possibly through increasing anti-oxidant enzyme activities, and may have a interaction with NO.

Physiological and transcriptional responses of broad bean ( Vicia faba L.) leaves to aluminium stress

Aluminium (Al) toxicity is the major factor-limiting crop productivity in acid soils. In the present study, physiological and transcriptional responses of broad bean leaves to Al stress were investigated. Malondialdehyde (MDA) content, H 2 O 2 content and protein carbonyls (PC) level in leaves were increased after 100 μ m AlCl 3 stress treatment, whereas the total protein content was decreased, compared with the plants without Al treatment. Stomatal closure in leaves of broad bean was increased after Al stress, suggesting that the photosynthesis rate might be affected by Al stress. The relative citrate secretion in leaves was decreased after Al treatment for 24 h according to the 13 C-NMR analysis, indicating that citrate in leaves might be transported to the root to chelate Al 3+ . To investigate the molecular mechanisms of Al toxicity in leaves of broad bean, a suppression subtractive hybridization (SSH) library was constructed to identify up-regulated genes: cDNA from leaves subjected to 12, 24, 48 and 72 h of 50 and 100 μ m AlCl 3 stress were used as testers and cDNA from leaves subjected to 0 μ m AlCl 3 treatment for the same lengths of time as above were used as a driver. The SSH analysis identified 156 non-redundant putative Al stress-responsive expressed sequence tags (ESTs) out of 960 clones. The ESTs were categorized into ten functional groups, which were involved in metabolism (0·21), protein synthesis and protein fate (0·10), photosynthesis and chloroplast structure (0·09), transporter (0·08), cell wall related (0·06), signal transduction (0·05), defence, stress and cell death (0·05), energy (0·03), transcription factor (0·03) and unknown proteins (0·30). The effect of Al treatment on expression of 15 selected genes was investigated by reverse transcription polymerase chain reaction (RT–PCR), confirming induction by Al stress. The results indicated that genes involved in organic acid metabolism, transport, photosynthesis and chloroplast structure, defence, stress and cell death might play important roles under Al stress.

Identification and evaluation of excess nitrate-responsive genes in celery (Apium graveolens L.) roots

Summary Salinity negatively impacts plant growth and productivity, but little is known about nitrate stress responses in celery (Apium graveolens L.). To improve our understanding of the adaptation of celery in nitrate-rich environments, we constructed a suppression-subtractive hybridisation (SSH) library. cDNAs from celery root tissue exposed to 100 mM total nitrate for 2, 4, 8, 24, 48, or 72 h were used as testers, and cDNAs of roots exposed to 0 mM nitrate for the same treatment times were used as drivers. Sequence analysis of the SSH cDNA library generated 202 high-quality expression sequence tags (ESTs), of which 112 were non-redundant. Eighty ESTs showed similarities to proteins of known function, and could be categorised into 11 functional groups, including metabolism, protein synthesis, cell defense and rescue, signal transduction, transport facilitation, genetic information processing, energy, cell structure, transcription, unknown, and no hits. Ten of the ESTs were selected by reverse transcription (RT)-PCR and their induction by nitrate stress was confirmed. An analysis of the physiological changes in celery roots indicated that hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels increased rapidly under the 100 mM total nitrate stress treatment. The expression of genes encoding peroxidase (POD), glutamine synthetase (GS), and a proline-rich protein (PRP) were up-regulated. The maximum activities of POD and GS, and free proline (Pro) contents were found 48 h and 72 h after 100 mM nitrate treatment. Collectively, these results contribute to our understanding of the molecular mechanism(s) of nitrate tolerance in celery, and possibly in other species.