Edwards G E, Robinson S P, Tyler N J, Walker D A
Department of Botany, The University of Sheffield, Sheffield S10 2TN, England.
Plant Physiol. 1978 Aug;62(2):313-9. doi: 10.1104/pp.62.2.313.
Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O(2) evolution of about 100 to 150 micromoles of O(2) per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO(3) with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O(2) per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5'-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5'-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO(3) and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.
从小麦（普通小麦）原生质体分离得到的原生质体、原生质体提取物（完整叶绿体加叶绿体以外的物质）和叶绿体，在20℃和饱和碳酸氢盐条件下，通过光依赖的氧气释放来测定光合作用速率，每毫克叶绿素每小时约为100至150微摩尔氧气。该活性所需的测定条件为：对于原生质体，0.4摩尔山梨醇、50毫摩尔N-2-羟乙基哌嗪-N'-2-乙磺酸KOH（pH 7.6）和10毫摩尔碳酸氢钠；对于原生质体提取物和叶绿体，还需要1至10毫摩尔乙二胺四乙酸（EDTA）和0.2至0.5毫摩尔无机正磷酸盐（Pi）。在光合作用很大程度上依赖外源Pi之前，原生质体提取物每毫克叶绿素释放约6微摩尔氧气，而叶绿体的光合作用从一开始就对外源Pi有更强的依赖性。在最佳Pi水平下，6日龄小麦植株的叶绿体光合作用与添加5毫摩尔无机焦磷酸（PPi）加0.2毫摩尔腺苷-5'-二磷酸（ADP）时相似。单独添加PPi或ADP均抑制光合作用。当叶绿体在黑暗中孵育2至6分钟时，5毫摩尔PPi强烈抑制光合作用，而加入腺苷-5'-三磷酸（ATP）或ADP（0.2至0.6毫摩尔）可解除这种抑制。9日龄小麦叶片的叶绿体对PPi抑制的敏感性稍低，对ADP几乎没有抑制作用。从原生质体分离并在光照前添加0.3毫摩尔Pi进行测定的叶绿体，其诱导时间从不到1分钟到16分钟不等，这取决于分离后测定的时间和培养基成分。为了获得最大光合作用速率和最短诱导时间，在叶绿体分离、重悬和测定培养基中需要碳酸氢钠和螯合剂EDTA或PPi（+ATP）。在分离和重悬培养基中加入这些物质后，叶绿体在冰上储存的前20至30分钟内，诱导时间迅速缩短。本文讨论了从小麦原生质体中分离完整且具有光合功能的叶绿体的要求。
