Identification and Preliminary Characterization of Wheat Mesophyll Cells 65 kDa Microtubule-associated Protein

[Objective] This study aimed to test the characteristics of MAP65 in wheat mesophyll cells by co-sedimentation experiments. [Method] We used wheat Linyou 2018 as the experimental material. Western blotting analysis was carried out to de- tect the existing of 65 kDa MAP in the wheat mesophyll cells; spectrophotometery and SDS-PAGE were adopted to analyze the role of MAP65 in microtubules poly- merization in vitro. [Result] MAP65 indeed existed in wheat mesophyll cells. Co-sedi- mentation experiments showed that MAP65 can bind to microtubules, with the basic characteristics of the microtubule-associated proteins. Turbidimetric experiments showed that microtubule polymerization depended on the concentration of MAP65. Low concentrations of MAP65 promoted microtubule polymerization while high con- centrations of MAP65 inhibited microtubule polymerization. [Conclusion] This study laid significant basis for further research on physiological function of MAP65 protein.

Effects of High Temperature Stress on Microscopic and Ultrastructural Characteristics of Mesophyll Cells in Flag Leaves of Rice

The microscopic and ultrastructural characteristics of mesophyll cells in flag leaves of two rice lines （a thermosensitive line 4628 and a thermo-resistant line 996） under high temperature stress （37℃ during 8：00-17：00 and 30℃ during 17：00-8：00） were investigated using an optical and a transmission electron microscopy. The membrane permeability and malondialdehyde content increased under the high temperature stress, and the increase of both variables was greater in the line 4628 than in the line 996. Under the high temperature stress, the line 996 showed tightly arranged mesophyll cells in flag leaves, fully developed vascular bundles and some closed stomata, whereas the line 4628 suffered from injury because of undeveloped vascular bundles, loosely arranged mesophyll cells and opened stomata. The mesophyll cells in flag leaves of the line 4628 were severely damaged under the high temperature stress, i.e. the chloroplast envelope became blurred, the grana thylakoid layer was arranged loosely and irregularly, the stroma layer disappeared, many osmiophilic granules appeared within the chloroplast, the outer membrane of mitochondria and the nucleus disintegrated and became blurred, the nucleolus disappeared, and much fibrillar-granular materials appeared within the nucleus. In contrast, the mesophyll cells in flag leaves of the line 996 maintained an intact ultrastructure under the high temperature stress. From these results, it is suggested that the ultrastructural modification of the cell membrane system is the primary plant response to high temperature stress and can be used as an index to evaluate the crop heat tolerance.

Effects of Exterior Abscisic Acid on Calcium Distribution of Mesophyll Cells and Calcium Concentration of Guard Cells in Maize Seedlings

In this study, the direct effects of exterior abscisic acid （ABA） on both calcium distribution of mesophyll cells and cytosolic calcium concentration of guard cells were examined. The distribution of Ca^2＋ localization were observed with calcium antimonate precipitate-electromicroscopic-cyto-chemical methods after treated with ABA and pretreated with ethylene glycol-bis-（2-aminoethylether）-N,N,N＇,N＇-tetraacetic acid （EGTA）, verapamil （Vp）, and trifluoperazine （TFP）. The laser scanning confocal microscopy was used to measure the cytosolic calcium concentrations of guard cells under different treatments. The results showed that the cytosolic Ca^2＋ concentration of mesophyll ceils was induced to increase by ABA, but to decrease in both outside cell and the vacuoles within 10 rain after treatments. The cytosolic calcium concentration of guard cells was increased gradually with the lag in treatment time. However, both EGTA and TFP could inverse those effects, indicating that the increase of cytosolic calcium induced by exterior ABA was mainly caused by calcium influx. The results also showed that calmodulin could influence both the calcium distribution of mesophyll cells and calcium concentration of guard cells. It shows that calmodulin participates in the process of ABA signal transduction, but the mechanism is not known as yet. The changes both calcium distribution of mesophyll cells and calcium concentration of guard cells further proved that the variations of cytosolic Ca^2＋ concentration induced by ABA were involved in the stomatal movements of maize seedlings.

Characteristics of the Mesophyllous Cells in the Sheaths of Rice (Oryza sativa L.)

The photosynthesis of rice sheath plays a significant role to furnish rice yield, and it is accounted for 10 to 20% of the final yield. But, limited studies have been done to address this phenomenon and to characterize the mesophyllous cells of rice sheath and how it may attribute to the rice yield. In this paper, super hybrid rice Liangyoupeijiu, its parents Wumang 9311 and Peiai 64S, and hybrid rice Shanyou 63 were studied as the experimental materials, and the characteristics of the mesophyllous cells of rice sheaths were examined by microscopic and super-microscopic observation as well as chlorophyll absorption spectrums. The results showed that rice sheath was rich in the intact mesophyllous cells full of chloroplasts, grana and thylakoids, which were much the same as those of rice blade. The absorption spectrum curves of the Chl. a and b of the sheaths were similar to those of the blades. The stomatal density in the outer epidermises of the sheaths was comparable to those in the up- and down-epidermises of the blades. The significant tests proved that the amount of chloroplast per mesophyllous cell of the sheaths was almost the same as those of the blades, and the mesophyllous cells in the sheaths were also rich in chlorophylls. The chlorophyll content of rice sheath reached about 50% of the chlorophyll content of rice blade, and the P, of the sheath/the blade ranged from 13.60 to 34.57%. Therefore, rice sheath was also full of the intact photosynthetic apparatus similar to those in rice blade, and had capabilities of photosynthesis. The statistical analysis revealed that the physiological senescence of the photosynthetic apparatus in both the sheath and the blade of Liangyoupeijiu was significantly slower than those of the other varieties at the late stages. The profuse grain-filling stage was an inflexion point of the physiological senescence of the chloroplasts and the chlorophylls of both the blades and the sheaths.

Gas flaring cause shifts in mesophyll and stomatal functional traits of Betula pubescens Ehrh.

In petroleum-producing territories of West Siberia(Russia),oil well gas flares have a thermal effect on nearby plant communities.Such communities can be used as models for studying plant acclimation to global warming.In the present study on the effect of the hydrothermal regime at the flare sites on mesophyll and stomatal functional traits of Betula pubescens,leaves were collected from trees at250 m(control site[CS]),200,150 and 100 m(maximum impact site[MIS])from a flare.From the CS to MIS site,the average annual air temperature increased by 0.5℃and bog water level decreased by 17 cm.On plants from the MIS,stomata were 16%smaller and density was 20%lower compared to those at the CS,resulting in lower maximum stomatal conductance in plants from the MIS(mean±SE:MIS 0.84±0.05 mol·m^(-2)s^(-1),CS 1.24±0.06 mol·m^(-2)s^(-1);F=12.6,P<0.01).Mesophyll cell volume was 1.9 times lower at MIS than at CS.Chloroplast numbers per cell also declined with distance from the flares,from 21(MIS)to18(CS;F=15.6,P<0.001),and chloroplast volume was 24%higher at the CS,whereas the number of mesophyll cells and chloroplasts numbers per unit leaf area were 1.9 and 1.8 times higher at the MIS than at the CS,respectively.As a result,leaves from the MIS had a large total mesophyll cell(Ames/A)and chloroplast(Achl/A)surface area per unit leaf area,resulting in a 46%increase in mesophyll conductance in plants from the MIS.Thus,structural changes in leaf epidermis consisted of a decrease in stomatal size and number,could lower transpiration losses with higher temperatures and less water.To compensate for the reduction in leaf conductance due to a decrease in stomatal conductance under these conditions,an increase in the number of mesophyll cells and chloroplasts per unit area provides a greater gas-exchange area and mesophyll conductance.

Cytochemical localization of ATPase and sub-cellular variation in mesophyll cell of Cyclocarya paliurus seedlings under iso-osmotic stress and calcium regulation

The ultrastructural distribution and active location of ATPase and the ultrastructural variations were investigated in mesophyll cells of Cyclocarya paliurus seedlings after iso-osmotic salt/water treatments in combination with calcium regulation. C. paliurus seed- lings were treated with five groups （control, 85 mM NaCl, 85 mM NaCl＋ 12 mM Ca（NO3）2, PEG iso-osmotic to 85 mM NaCl and PEG iso-osmotic to 85 mM NaCl＋12 mM Ca（NO））2） in a hydroponic system in a phytotron. Results show that under normal growth conditions the ATPase activity was low and the enzyme was primarily located on the nucleus. After 12 days of iso-osmotic salt/water treatments, ATPase activity on the tonoptast increased. Osmiophilic globules for iso-osmotic water treatment were greater than that for iso-osmotie salt treatments. The ATPase activity increased and was mostly transferred onto the nucleus for calcium regulation treatment under iso- osmotic salt/water stresses, and the osmiophilic globules significantly decreased under iso-osmotic water stress with calcium regulation. The ATPase located on the nucleus indicated that the degree of salt/drought damage that seedlings suffered was slighter, while the amount of the enzyme located on the tonoplast showed that the degree of salt/drought damage there was more serious. After 4 and 20 days of iso- osmotic treatments, the injury suffered by the leaf ultrastructures of C. paliurus seedlings for iso-osmotic treatment with calcium regula- tion was lower than those without calcium regulation, especially for the iso-osmotic water treatments. Preliminary analysis suggests that the iniury suffered by C. paliurus seedlings was lower for iso-osmotic salt treatments than for iso-osmotic water treatments, while the effect of calcium regulation under iso-osmotic water stress was greater than that of the iso-osmotic salt stress.

PCR Amplification，Cloning and Sequencing of RbcL Coding Region in Mesophyll Cell and Bundle Sheath Cell of Sorghum（Sorghum bicolor L．）

The Primacy question addressed in our study is: Is the difterntial expression of rbcL gene in mesophyll cells and in bundle sheath cells related to the sequence of the gene per se?An enzymatic approach was fist established to separate the two groups of cells. Microscopic examination revealed satisfactory separation effect: minimal mutual contamination was found so that no mistake might be introduced into biochemical or molecular biological expeitments using such preparations. CpDNA were isolated from mesophyll cells and from bundle sheath cells and coding region of rbcL gene was obtained from each by PCR ampilfication.Cloning and sequencing were then done on them.Compartive analysis , however, revealed identical sequence, with a length of 1,368 bp, encoding 456 amino acids. Since sequences of the non-coding regions of rbcL gene in masephyll sad bundle sheath have not been obtained, it can not yet be concluded that the differential expression is not related to the sequence itself. Nevertheless,It sesems justifiable to infer that whatever difference there may be between the sequences of rbcL gene in two groups of cells can only be found in the non-coding regions(including promoter and the 3' down stream region).

Effects of light intensity on leaf microstructure and growth of rape seedlings cultivated under a combination of red and blue LEDs

The aim of this study was to evaluate the growth of rape （Brassica napus L.） seedlings under different light intensities to select appropriate conditions for cultivation in an indoor system. Seedlings were grown under different light intensities of red and blue light provided by light-emitting diodes （LEDs） and their self-adjustment ability and changes in leaf microstructure were evaluated. Light was supplied by red LEDs with peak wavelengths of 630 （R1） and 660 nm （R2） and by blue LEDs （B） with a peak wavelength of 445 nm （the light intensity ratio of R1：R2：B was 3：3：2）, at intensities of 400 （R1R2B400）, 300 （R1R2B300）, and 200 μmol m-2 s-1 （R1R2B200）. Natural solar light served as the control （C）. Plant height, stem diameter, root length, leaf area, and dry weight of rape seedlings gradually increased with increasing light intensity. The seedlings in the R1R2B400 treatment grew more vigorously, while those in the R1R2B200 treatment were weaker. The photosynthetic pigment contents did not differ significantly between the R1R2B400 treatment and C, but were significantly lower in the R1R2B300 and R1R2B200 treatments. The highest intercellular CO2concentration, stomatal conductance, and transpiration rate were in the R1R2B300 treatment. The highest photosynthetic rate was in the R1R2B400 treatment, and was related to more compact leaves, thicker and tidier palisade and spongy tissues, and well-developed chloroplasts. In contrast, the seedlings in the R1R2B200 treatment had disordered mesophyll cells, round chloroplasts, and fractured and fuzzy grana lamellae, all of which inhibited plant growth. In conclusion, the seedlings in the R1R2B400 treatment had well-developed leaves, which favored photosynthesis. Compared with the light intensities below 300 μmol m-2 s-1, the light intensity of 400 μmol m-2 s-1 provided by a cembination of red and blue LEDs was beneficial for cultivating strong and healthy rape seedlings in an artificial system.

Foliar application of zinc promotes cadmium absorption by increasing expression of cadmium transporter genes and activities of antioxidative enzymes in winter wheat

Cadmium(Cd)is a deleterious non-essential metal in plants.To elucidate the mechanisms by which zinc(Zn)application alleviates cadmium(Cd)toxicity in wheat,we characterized plant growth,antioxidant system,leaf cell ultrastructure,and Cd transporter gene expression in winter wheat under Cd exposure(50μmol L^(-1)Cd)with foliar Zn application in a hydroponic experiment.Results showed that Zn addition(Zn+Cd)or pretreatment(pre-Zn+Cd)at 2 g L^(-1)as Zn_(S)O_(4)·7H_(2)O significantly exacerbated Cd-induced growth inhibition and diminished root morphological parameters,root cell viability,and chlorophyll content.In addition,the Cd content increased in roots and shoots in the Zn+Cd and pre-Zn+Cd treatments,but the Cd translocation factor decreased,when compared to the treatment without Zn application.After Zn application,the Cd content increased in the root cell wall fraction but decreased in the soluble fraction.The activities of antioxidative enzymes(superoxide dismutase,peroxidase,and catalase)and the contents of non-enzymatic antioxidants(malondialdehyde,ascorbic acid,and glutathione)significantly increased in the roots and shoots of wheat after Cd exposure,particularly in the Zn+Cd and pre-Zn+Cd treatments.Gene expression analysis showed that five genes(TaHMA2,TRIAE5370,TCONS1113,TRIAE5770,TRIAE1060,and TCONS5200)participated in root absorption of Cd,whereas TCONS5200 and TRIAE5660 contributed to Cd transfer to shoots.Foliar application of Zn increased the number of chloroplasts,but the chloroplast structure was destroyed in the Zn+Cd treatment.These results indicated that 2 g L^(-1)ZnSO4·7H2O increased the toxicity of 50μmol L^(-1)Cd.Increased Cd uptake due to the upregulated expression of Cd transporter genes and increased reactive oxygen species accumulation may be the reasons why Zn application aggravated Cd toxicity.