Effects of Salinity Stress on the Structure of Bundle Sheath and Mesophyll Chloroplasts in NAD-Malic Enzyme and PCK Type C4 Plants

Abstract

The effect of NaCl stress on the structure of leaf chloroplasts was investigated in several NAD-Malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) type C₄ plant species. Seedlings of the monocot species, except Zoysia japonica, grown in 300 mL pots were subjected to salt stress by adding 50 mL of 3% NaCl solution per day to the soil for 5 d after the fourth leaf blades were fully developed. Z. japonica and the dicot species, Amaranthus tricolor, were also treated with 3% NaCl in a similar manner from 5 wk after germination. Salt stress negatively affected the growth, chlorophyll content and chloroplast structure in all the species. At the ultrastructure level, swelling of thylakoids and disruption of envelopes were more or less observed in mesophyll cell (MC) chloroplasts after salt treatment. The structure of bundle sheath cell (BSC) chloroplasts, on the other hand, was hardly damaged under salt condition although stromal and starch areas were considerably decreased. Furthermore, salinity induced granal development in BSC chloroplasts in most species; the number of thylakoids per granum, granal indices and appressed thylakoid density in salt-treated plants were generally higher than those in control. Since the similar responses have also been reported in all NADP-ME type C₄ species investigated in our previous study, the high sensitivity to salt stress in MC chloroplasts and the granal development in BSC chloroplasts by salinity were considered to be common phenomena in all three C₄ subtypes.     

Gene Expression and Accumulation of Rubisco in Bundle Sheath and Mesophyll Cells during Leaf Development and Senescence in Rice,a C3 Plant

Abstract

Gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (rbcL) and small subunit (rbcS) in bundle sheath and mesophyll cells of rice, a C₃ plant, was examined during leaf development and senescence by in situ hybridization. Localization of Rubisco protein in both cells was also examined by immuno-electron microscopy. Gene expression and accumulation of Rubisco were related with the chlorophyll fluorescence parameters. The chlorophyll fluorescence parameters, such as F_v/F_m and Φ_psii, gradually increased during leaf development with the increase in the accumulation of Rubisco. However, the chlorophyll fluorescence parameters decreased earlier than the Rubisco content during leaf senescence. The expression of rbcS decreased earlier in bundle sheath cells than in mesophyll cells during leaf development, whereas the expression of rbcL in both cells was retained during leaf development and decreased during leaf senescence. On the other hand, Rubisco content of bundle sheath and mesophyll cells increased during leaf development and decreased during leaf senescence. Rubisco was retained even after the disappearance of the expression of rbcS and rbcL detectable by in situ hybridization. The present results suggest that the expression pattern of rbcS in bundle sheath cells was somewhat different from that in mesophyll cells, but this difference was not reflected in Rubisco content.     

玉米C4光合叶不同部位“花环”结构及叶绿素含量的变化

以玉米进行C4光合的全展第5位叶片为材料,分析从叶基部到顶部的解剖结构和叶绿素含量变化,研究玉米C4光合叶片"花环"结构随叶片发育的变化规律。结果表明,玉米第5位叶从基部到顶部都具有完整的典型"花环"结构,维管束鞘细胞(BSC)和叶肉细胞(MC)的体积在叶片发育过程中具有渐变性,从叶基部到顶部BSC和MC均呈先增大后变小的趋势,而且叶绿素a、b和a+b含量呈相同的变化趋势,说明BSC和MC细胞体积与叶绿素含量的变化具有相关性。叶绿素a/b总体呈上升趋势,说明玉米第5叶基部到顶部的光合途径存在C3向C4转变的过程。玉米第5叶不同部位C4光合途径发育的渐变性比前3叶更为明显。     

Photosynthetic Characteristics of an Amphibious C4 Plant,Eleocharis retroflexa ssp. chaetaria

Abstract

Eleocharis retroflexa (Poir.) Urban ssp. chaetaria (Roem. & Schult.) T. Koyama, an amphibious leafless sedge, grows not only under terrestrial conditions but also under completely submerged aquatic conditions. We investigated the photosynthetic traits and structural features of the culms, which are the photosynthetic organs, in the terrestrial and submerged forms of this species and compared them with those of other amphibious species of Eleocharis which are known to change the photosynthetic modes. The culms of the terrestrial form had Kranz anatomy with well-developed Kranz (bundle sheath) cells and high levels of C₄ enzyme activity typical of the NAD-malic enzyme (NAD-ME) subtype of C₄ metabolism. They also had a δ ¹³C value typical of C₄ plants, indicating that the terrestrial form fixes carbon through the C₄ pathway. The culms of the submerged form had not only a Kranz-like anatomy but also revealed anatomical traits typical of leaves of submerged aquatic plants. The activities of the C₄ enzymes in the submerged form were lower than those in the terrestrial form, but were still in the range typical of G4 plants, ¹⁴C pulse-¹²C chase experiments with the submerged form indicated that almost all of the fixed ¹⁴C was incorporated into G4 compounds, and subsequently the raioactivity was transferred into C₃ compounds and sucrose. The submerged form showed no diurnal fluctuation in malate level. These data demonstrate that a C₄ metabolism is operative even in the submerged form. This unique amphibious C₄ plant provides an intriguing example of the physiological and ecological adaptability of C₄plants.     

甘肃省玉米鞘腐病病原菌鉴定及生物学特性观察

2021年6月在甘肃省张掖市甘州区党寨镇自然感玉米鞘腐病的玉米植株中分离鉴定病原菌并观察分析病原菌的生物学特性。结果表明,该致病菌株（QF-1RC）形态学结构与拟轮枝镰孢菌（Fusarium verticillioides）一致。基于rDNA-ITS和微管蛋白序列分析发现,该病原菌与拟轮枝镰孢菌MN652651和FR997663的相似度达99%,结合形态学和与分子生物学的鉴定结果将其定为拟轮枝镰孢菌。生物学特性观察发现,菌丝生长最适温度为25℃,弱酸弱碱环境下均有利于菌丝生长,全光照环境条件下菌丝生长速率快,不同C、N源对菌丝生长和产孢量有一定的影响,D-纤维二糖培养基上菌丝生长相对受限,在海藻糖和D-半乳糖培养基上菌株产孢量较多,硫酸铵培养基上病原菌生长相对受限,乙酸铵培养基上菌株产孢量最多,硝酸钠培养基上菌株产孢量较少。     

NaCl和Na2SO4胁迫对玉米幼苗渗透调节物质含量的影响

研究了NaCl和Na₂SO₄胁迫对玉米幼苗电解质渗漏、根系活力及脯氨酸、可溶性糖、可溶性蛋白等渗透调节物质含量的影响。结果表明，玉米在NaCl作用下其电解质渗漏、根系活力及渗透调节物质含量的变化和Na₂SO₄胁迫相似，电解质渗漏、根系活力及渗透调节物质含量均随着盐浓度的增加而加剧。NaCl胁迫下增幅大于Na₂SO₄胁迫，表明NaCl胁迫对玉米幼苗的伤害大于Na₂SO₄胁迫。     

Ultrastructure of Fusion Product between Protoplasts from C3 and C4 Species of Amaranthaceae

Summary

This paper describes the ultrastructure of the electric field-induced fusion products of C₃ and C₄ species of Amaranthaceae at the early developmental stage. Protoplasts of C₃ species were isolated from a Ceiosia cristata L. cell suspension and, those of C₄ species were isolated from an Amaranthus tricolor L. cotyledon. Incompatibility occurred in the C₃/C₄ hybrid. The incompatibility reactions were detected in the newly formed hybrid cells accompanied with significant changes in the nucleolus (segregation of nucleolar components) and plastids (cup-like shape or amoeboid plastid enclosing cytoplasmic materials) of C₃ species parent. The structural changes in the organelles of the C₄ partner were less marked. After 5 days of culture, most organelles showed high cellular activity, and a normal dedifferentiation process of mesophyll chloroplasts was observed. At this stage nucleolar segregation was not detected and the C₃ species plastids were difficult to distinguish from the proplastids formed from mesophyll chloroplasts. In addition, some mitochondria showed bursting-like structure. However, under the culture condition used these somatic incompatibility did not seem to impair further growth of fusion products since they were still proliferating well resulting in callus formation.     

玉米C_4光合酶基因导入对拟南芥光合特性及抗旱性的影响

为了解玉米C_4型光合酶基因对C3植物拟南芥光合特性的影响及其对干旱胁迫的响应,分别以过表达ZmPEPC(磷酸烯醇式丙酮酸羧化酶)、ZmPPDK(丙酮酸磷酸二激酶)和ZmNADP-ME(依赖于NADP的苹果酸酶)单个酶基因的拟南芥株系(分别简写为PC、PK、ME),以及过表达PEPC+PPDK和PPDK+NADP-ME两个酶基因的拟南芥株系(分别简写为PCK、PKM)为供试材料,在开花期停止浇水,并分别于干旱胁迫处理前1天、第5天、第10天和结束干旱胁迫复水处理5d时测定转基因拟南芥中目标基因的表达量、光合速率、水分利用效率和酶活性。结果表明,在正常生长条件下,PCK类型株系的PEPC酶活性、PPDK酶活性、净光合速率(Pn)以及水分利用效率(WUE)较野生型拟南芥分别高52%、20%、24%和55%,除PPDK酶活性外,PCK类型株系各指标测定值的增幅均高于其他转基因类型株系,综合表现最优。不同类型株系的上述测定指标总体表现为PCKPCPK、PKMME。干旱胁迫处理5d时,各类型株系中目标基因的表达量、光合酶活性、Pn和WUE均有所上升。干旱胁迫处理10d时,拟南芥受到严重损伤,上述指标的测定值均下降。复水5d时上述指标得到不同程度的恢复,不同类型转基因株系的各项测定指标在不同干旱胁迫处理中总体仍表现为PCK最优,PC次之。各转基因株系均优于受体非转基因拟南芥。     

Ultrastructure of Hybrid Callus between C3 and C4 Species of Amaranthaceae

Summary

Unlike the parent line, the hybrid calli between C₃ and C₄ species of Amaranthaceae have poor division capability. Therefore, in this study the fine structures of hybrid callus derived from protoplasts of Celosia cristata L. cv. Pink Charm (C₃ species) cell suspension and Amaranthus tricolor L. cv. Perfecta (C₄ species) cotyledon callus were investigated by light and electron microscopy, and compared with the parental. All callus lines were composed of parenchymatous cells possessing a voluminous vacuole. Parental callus lines contained organelles with a relatively normal structure. The peculiar feature characterizing the C₃/C₄ hybrid callus was the presence of highly lobed nucleus with multinucleoli and numerous small vacuoles having autophagic activity scattered in the cytoplasm. The autophagic activity is apparently related to the existence of several inclusions such as cytoplasmic organelles in the central vacuole. It might also be related to the pronounced expansion of central vacuole and the reduced cytoplasm. The failure to sustain proliferation related to some atypical features of the organelles is discussed.     

Rubisco small subunits of C4 plants,Napier grass and guinea grass confer C4-like catalytic properties on Rubisco in rice

ABSTRACT

Overexpression of Rubisco small subunit (RbcS) of C₄ plant, sorghum (sorghum bicolor) was shown to enhance the catalytic turnover rate (k _cat) of Rubisco in rice (Oryza sativa). In this study, the effects of other Rubisco small subunits of C₄ plants, Napier grass (Pennisetum purpureum) and guinea grass (Megathyrsus maximus) on kinetic properties of Rubisco in rice were studied. The expression levels of Napier grass RbcS (NgRbcS) and guinea grass RbcS (GgRbcS) proteins accounted for 41% and 45% of total RbcS, respectively in homozygous overexpression lines. The k _cat and K _m for CO₂ (Kc) of Rubisco were increased in all transgenic lines. Interestingly, the k _cat was markedly higher in NgRbcS homozygous line, whereas K _c was notably higher in GgRbcS homozygous line. Although its effects depend on species, these results suggest that the introduction of C₄ RbcS are effective approaches to alter the catalytic properties of Rubisco in rice.     

玉米株型相关基因ZmDwarf4的克隆及表达分析

以松散型玉米自交系沈137为材料,利用同源克隆法成功克隆与水稻Dwarf4基因同源的玉米Dwarf4基因。序列分析发现,该基因cDNA序列全长1 626 bp,开放阅读框1 521 bp,编码506个氨基酸,与高粱、水稻、拟南芥的氨基酸同源性分别为95%、89%和71%。Real-Time PCR分析表明,ZmDwarf4基因的表达量为3～11叶期表达量逐渐增加,9～11叶期达到最大量,12～19叶期表达量逐渐降低,推测ZmDwarf4基因正向调控玉米叶夹角的大小。ZmDwarf4基因的表达量在叶片中最高,在叶枕中最低。不同激素处理的结果表明,用植物激素（IAA）处理的样品ZmDwarf4基因的表达量始终高于对照;用Brassinolide（BR）处理的样品6叶期ZmDwarf4基因的表达量高于对照,7～10叶期该基因的表达趋势与对照基本一致,但始终低于对照;用IAA+BR处理的样品,该基因的表达趋势与对照基本一致,但高于对照。ZmDwarf4参与玉米BR生物合成途径,进而调控其相关株型建成。     

低温胁迫下外源CaCl2对玉米种子萌发及幼苗生长的影响

以杂C-546为试材,采用10、20、40、80、160 mmol/L外源CaCl_2浸种和叶面喷施不同浓度处理种子,比较分析低温胁迫下玉米种子发芽率、发芽势、发芽指数和幼苗叶片相对电导率、可溶性蛋白、Pn、Fv/Fm、POD和SOD活性等生理生化指标的变化。结果表明,在低温抑制玉米幼苗生长条件下,一定浓度CaCl_2(10~80 mmol/L)处理可降低玉米幼苗生长伤害程度。80 mmol/L CaCl_2浸种处理对玉米低温胁迫的缓解效果最佳,与对照比较,发芽率提高8%,发芽势提高24.6%,发芽指数提高33.2,相对电导率降低51.8%,可溶性蛋白含量增加76.3%,SOD活性增加35.7%。适宜浓度的CaCl_2在低温条件下可促进玉米种子的萌发,降低低温对玉米幼苗造成的伤害。     

Structural and physiological responses of the C4 grass Sorghum bicolor to nitrogen limitation

Nitrogen (N) is one of the major nutrients influencing photosynthesis and productivity of C₄ plants as well as C₃ plants. C₄ photosynthesis operates through close coordination between mesophyll (M) and bundle sheath (BS) cells. However, how the development of structural and physiological traits in leaves of C₄ plants is regulated under N limitation remains uncertain. We investigated structural and physiological responses of leaves of the NADP-ME-type C₄ grass Sorghum bicolor to N limitation. Plants were grown under four levels of N supply (.05 to .6 g N per 5-L pot). Decreasing N supply resulted in decreases in net photosynthetic rate, stomatal conductance, leaf N and chlorophyll contents, and the activity ratio of phosphoenolpyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase and increases in δ¹³C values and photosynthetic N use efficiency. Low-N leaves were thinner and had smaller photosynthetic cells, especially in M, resulting in lower M/BS tissue area ratio, and contained smaller and fewer chloroplasts. The BS chloroplasts in the low-N leaves accumulated abundant starch grains. The number of thylakoids per granal stack was reduced in M chloroplasts but not in BS chloroplasts. The low-N leaves had thicker cell walls, especially in the BS cells, which might be associated with less negative δ¹³C values, and fewer plasmodesmata in the BS cells. These data reveal structural and physiological responses of C₄ plants to N limitation, most of which would be related to cellular N allocation, light use, CO₂ diffusion and leakiness, and metabolite transport under N limitation.     

水分胁迫下玉米叶片光合作用、脂质过氧化及超微结构变化的关系

研究了不同耐旱性品种在不同生育时期水分胁迫下叶片光合作用和呼吸作用、脂质过氧化作用、膜系统伤害、叶肉细胞超微结构变化之间的关系。在各生育时期水分胁迫下，光合作用和呼吸作用强度下降，SOD活性降低，脂质过氧化作用增强，膜透性增加，叶肉细胞的细胞器和质膜遭到严重破坏。结果表明，水分胁迫下脂质过氧化作用增强是造成细胞内膜系统紊乱和伤害的原因，而超微结构的破坏造成光合作用和呼吸作用的降低是导致作物减产的生理因素。     

辽宁省玉米穗腐病致病层出镰孢菌生物学特性、致病力与产伏马毒素B1比较

层出镰孢菌Fusarium proliferatum是辽宁省玉米穗腐病致病镰孢菌之一。比较4株层出镰孢菌的生物学特性、致病力、伏马毒素生物合成基因的表达量和产伏马毒素能力。结果表明,供试层出镰孢菌适宜生长温度为25℃～30℃,Fp-2、Fp-3、Fp-6最适温度为25℃,Fp-7最适温度为28℃。适宜pH值为5～8,Fp-2、Fp-7的最适pH值为7;Fp-3耐碱性,最适pH值为8;Fp-6耐酸性,最适pH值为5。12 h光暗交替有利于菌丝生长,适宜生长培养基为PSA、PDA。不同菌株对碳氮源的利用存在差异,适宜碳源为可溶性淀粉和蔗糖,适宜氮源为亚硝酸钠、天冬氨酸和脯氨酸。4株层出镰孢菌均能产生毒素,菌株间产毒能力差异显著,菌株Fp-7产伏马毒素B1能力最强,为77.65 mg/kg。菌株间产毒基因的相对表达量也存在差异,致病力无明显差异。     

Variations in structural,biochemical, and physiological traits of photosynthesis and resource use efficiency in Amaranthus species (NAD-ME-type C4)

C₄ plants show higher photosynthetic capacity and productivity than C₃ plants owing to a CO₂-concentrating mechanism in leaves, which reduces photorespiration. However, which traits regulate the photosynthetic capacity of C₄ plants remains unclear. We investigated structural, biochemical, and physiological traits associated with photosynthesis and resource use efficiency in 20 accessions of 12 species of Amaranthus, NAD-malic enzyme-type C₄ dicots. Net photosynthetic rate (P_N) ranged from 19.7 to 40.5 μmol m^?2 s^?1. P_N was positively correlated with stomatal conductance and nitrogen and chlorophyll contents of leaves and was weakly positively correlated with specific leaf weight. P_N was also positively correlated with the activity of the C₃ enzyme ribulose-1,5-bisphoshate carboxylase/oxygenase, but not with the activities of the C₄ enzymes phosphoenolpyruvate carboxylase and NAD-malic enzyme. Structural traits of leaves (stomatal density, guard cell length, leaf thickness, interveinal distance, sizes of mesophyll and bundle sheath cells and the area ratio between these cells) were not significantly correlated with P_N. These data suggest that some of the biochemical and physiological traits are involved in interspecific P_N variation, whereas structural traits are not directly involved. Photosynthetic nitrogen use efficiency ranged between 260 and 458 μmol mol^?1 N s^?1. Photosynthetic water use efficiency ranged between 5.6 and 10.4 mmol mol^?1. When these data were compared with previously published data of C₄ grasses, it is suggested that common mechanisms may determine the variations in resource use efficiency in grasses and this dicot group.     

Presence of chloroplasts in mestome sheath cells of the C3 Pooid grass Elymus tsukushiensis

ABSTRACT

The successful introduction of the C₄ pathway into C₃ crops would increase photosynthetic rates and crop productivity. However, our poor understanding of how Kranz leaf anatomy develops poses a great obstacle. In particular, the origin, development, and genetics of bundle sheath (BS) cells in C₄ plants are key points to elucidate. Here we report that Elymus tsukushiensis, a common C₃ grass of the subfamily Pooideae, contains chloroplasts in the mestome sheath (MS) cells of the leaf, unlike most MS cells of C₃ grasses. The chloroplasts are smaller than those of mesophyll cells. Immunogold localization showed that the chloroplasts and mitochondria of MS cells, respectively, accumulate ribulose 1,5-bisphosphate carboxylase/oxygenase and a photorespiratory enzyme, glycine decarboxylase, as in mesophyll cells. Thus, we suggest that the MS cells have weak photosynthetic and photorespiratory functions. This finding provides an insight into the development and evolution of C₄-type BS cells in leaves of C₃ grasses.     

玉米ZmKNOLLE基因的克隆及增强烟草耐盐性研究

从玉米基因组中克隆出ZmKNOLLE,对其蛋白的同源性和进化树进行分析,并在烟草中对其在盐胁迫中的功能进行验证。研究表明,SNARE蛋白参与植物抗逆性,研究描述了1个玉米SNARE蛋白ZmKNOLLE,其中,包含1个SNARE特定的结构域。它的c DNA为945bp,编码1个314个氨基酸的蛋白,预测分子量为34.54kD,等电点(p I)为7.02,在ZmKNOLLE蛋白C端有一个跨膜结构域。实时定量PCR结果显示,ZmKNOLLE能被盐和H_2O_2强烈诱导。通过农杆菌介导把ZmKNOLLE基因转化到烟草(W38)中,启动子为CaMV 35S。在盐胁迫下,与野生型相比,转基因烟草植株表现出较长的主根和较高的萌发率,相对含水量、POD、SOD活性较高,MDA含量较少,表明转基因烟草比野生型烟草对盐胁迫有更强的耐受性。     

Production of Raphanus sativus (C3)-Moricandia arvensis (C3-C4 intermediate) Monosomic and Disomic Addition Lines with Each Parentl Cytoplasmic Background and their Photorespiratory Characteristics

Abstract

We are maintaining five Moricandia arvensis monosomic addition lines of Raphanus sativus carrying R. sativus cytoplasm (autoplasmic MALs) and twelve M. arvensis MALs of R. sativus carrying M. arvensis cytoplasm (alloplasmic MALs) from BC₆ to BC₈ generation, and newly produced five M. arvensis disomic addition lines of R. sativus (autoplasmic DALs) and seven M. arvensis DALs of R. sativus carrying M. arvensis cytoplasm (alloplasmic DALs) from selfing and sib-crossing of the MALs and DALs in S₃BC₅ and S₂BC₆ generations. The structural, biochemical and physiological characteristics related to photorespiration of these MALs and DALs were compared to study the genetic mechanisms of the C₃-C₄ intermediate photosynthesis in the individual chromosomes of M. arvensis. The CO₂ compensation point of the autoplasmic and alloplasmic DALs (RMa-b and MaR-b DALs) with one pair of M. arvensis ‘b’ chromosome were 29.4 and 30.1 μmol mol-¹, respectively, which were significantly lower than that of other DALs and MALs as well as R. sativus (34.5 𰂼mol mol-¹). An immunogold electron microscopic study of the P-protein of glycine decarboxylase (GDC) in photosynthetic cells of the RMa-b DAL revealed that the bundle sheath cell (BSC) mitochondria were more intensively labeled for the protein than the mesophyll cell (MC) mitochondria. The ratio of the labeling density of the BSC mitochondria to that of the MC mitochondria was 1.13, which lies between values in M. arvensis (2.66) and R. sativus (0.76). These data suggest that the ‘b’ chromosome of M. arvensis genome controls the expression of C₃-C₄ intermediate characteristics.     

玉米田土壤中N_2O的释放特性及对不同因素的响应

采用土壤释放气体常压培养模拟装置,通过设置不同的氮肥施用量、土壤含水量、培养温度及土壤酸化等处理,明确N_2O释放特性及对环境因素的响应。结果表明,纯氮施入量由70 mg/kg增加至300 mg/kg,N_2O释放总量分别增加了32.9%(对照)和21.2%(酸化土壤);含水量由15%增加至25%;N_2O释放总量分别增加26.5%(对照)和16.1%(酸化土壤)。不同培养温度对比发现,N_2O释放总量变化趋势为25℃处理35℃处理15℃处理,25℃处理与15℃处理相比,N_2O释放总量增加了12.7%(对照)和20.0%(酸化土壤)。与对照处理相比,酸化处理N_2O释放总量平均增幅均高于5%。试验所设定的条件范围内,增施氮肥、提高含水量、土壤酸化导致N_2O释放强度增大,合适的培养温度有利于N_2O释放。