Breidenbach, R. William : R. William Breidenbach, Agronomist, Plant Growth Laboratory, Agronomy and Range Science, U.C., Davis

Goldberg, Robert B. : Robert B. Goldberg, Associate Professor, Biology, U.C., Los Angeles

Taylor, Scott E. : Scott E. Taylor, Post Graduate Research Plant Physiologist, Plant and Soil Biology, U.C., Berkeley.

ide , thereby providing a carbon source for a limiting factor in crop yield . Transforma - whether or not geneticists should continue to photosynthesis . It is found in the chloroplast tion of a plant with a gene encoding a more use the traditional methods of inducing and efficient RuBPCase might therefore improve and consists of eight small subunit polypep - recovering mutants in whole , complicated crop yield . Once the host plant has been tides encoded by a gene in the nucleus and sort plants . The answer will depend on what eight largesubunit polypeptidesencoded by a transformed with a cloned gene , however , it of mutant is desired and the ability of the is necessarythat this new gene be expressed in gene in the chloroplast . It has been known for biological assay system to permit recognition the correct quantity in the correct leaf cells . some time that light influencesthe amount of of it . When the phenomenon to benefit from Problems may arise RuBPCase in developing leaves of many if the cloned gene is mutational analysis involves sequential ac - plants . We have isolated synthetic DNA regulated by a different set of signals than of molecules compartmentalized in tion copies of small subunit mRNA ( cloned those employed in the host plant . It is clear space or time , or all but the most simple mo - cDNA ) using recombinant DNA technology . that attention must be paid to the details of lecular interactions , classical genetic tech - us a very sensitive a gene when it is passed into the This cloned cDNA gives regulation of nology is the cutting edge . For example , there probe with which we can detect small quanti - genome of a different plant . is little question that classical genetics would have the greater chance of finding a mutant ties of small subunit mRNA and measure William C . Taylor , Assistant Professor of Genet - altering the anaerobic gene program itself . changes in its relative concentration within ics and Assistant Geneticist ; Timothy Nelson , w ; American Cancer Society Postdoctoral Fell0 so little about the rules or Since we know the cell . We were unable to detect small sub - Mark Harpster , Lino Fragoso , and Belinda Mar - mechanism governing how development con - unit mRNA when seedlingswere grown in the tineau , graduate students , Genetics ; Judy Yama - trols groups of genes , it is wise to allow the complete absence of light . However , when guchi , Staff Research Associate , Genetics ; and Stephen Mayfield , graduate student , Plant Physi - organism maximum freedomto give us clues . seedlings that had been grown in the dark U.C . , Berkeley . ology . All are at Once particular DNA sequences are defined , to continuous illumination , were shifted in vitro genetics becomes the approach then there was a rapid and dramatic accumulation of choice . of small subunit mRNA . Light induces a of small greater than 1,000 - fold stimulation Michael Freeling , Associate Professor , Genetics , U.C . , Berkeley . mRNA synthesis in bean primary leaves . of atmospheric carbon dioxide is Fixation ( Zeamays , inbred line more complex in corn Storage protein genes B73 , donated by Pioneer Seed Company , Des Moines , Iowa ) . The processinvolvestwo dif - R . William Breidenbach ferent enzymes located in different cell types . Robert B . Goldberg Leaf protein synthesis of RuBPCase is restricted to the chloroplasts Scott E . Taylor vascular bundle sheath cells ; phosphoenol - pyruvate carboxylase ( PEPCase ) is found William C . Taylor only in the cytoplasm of mesophyll cells . We Timothy Nelson have measured the accumulation of both pro - Interest in the developmentaland molecular Mark Harpster a variety of dark and light growth teins under biology of the proteins that accumulateas re - Lino Fragoso regimens using sensitive antibody probes . serves in seeds has become keen in recent Belinda Martineau Synthesis of both proteins begins about four years . Although most plant cells contain large Steven Mayfield or five days after germination . This time of numbers of different proteins , each present Judy Yamaguchi synthesis initiation is independent of light . only in small quantities , food chemists , using Accumulation of both carboxylases proceeds criteria of size and solubility , long ago found rapidly , even in the absenceof light . The only that most of the protein in seeds of the soy - T h e most abundant proteins in the leaves measurable effect of light is to increase the to be com - bean and other legumes appears of higher plants perform specialized func - rate of accumulation of both enzymes . posed of only a few different kinds . In the of these pro - tions in photosynthesis . Many Contrasted to this light - independent regu - late 1960s we recognized that , if this were teins are located within the chloroplast . Some lation of PEPCase and RuBPCase during true , it was likely that a correspondingly are encoded by the chloroplast genome , and of the corn leaf developmentis the regulation smallnumber of different kinds of messenger of some by the nuclear genome.The synthesis chlorophyll a / b binding protein ( chl a / b RNA ( mRNA ) molecules encoded to direct several of these proteins has been shownto be protein ) . This protein forms a complex with the synthesis of the storage proteins would controlled by light . chlorophylls a and b and is responsible for also be present in higher concentrations in of these We are interestedin how synthesis harvestingthe light energy that drives photo - seed tissue cells . Higher concentrations of proteins is regulated during leaf develop - synthesis . Our preliminary studies indicate specific mRNAâ??s would make it much easier ment . Our studies have shown that environ - that light regulates synthesis of chl a / b pro - to learn how these intermediaries between mental factors interact with the leafâ??s devel - tein in corn . In the absenceof light there is no genes and their protein products are modu - opmental program to influence the quantity immunologically detectable chl a / b protein . lated and , in turn , how they control the rates of several abundant and timing of synthesis of leaf protein synthesisin corn Our studies of protein production . proteins . We have also discovered that syn - and in beans demonstrate that genes en - Thesetwo aspects of the regulationof gene thesis of the same protein is regulated in very coding the same protein can be regulated in expressionare among the most important un - different ways in two different plants . different ways in different plants . This fact , answered questions in biology and are funda - The most abundant protein in green leaves while interesting to the plant developmental of plant im - mental to the practical problems of the bean plant biologist , has profound implications for the ( Phaseolus vulgaris â?? Red provement by either conventional plant Kidney â?? ) is ribulose - 1 , 5 - bisphosphate car - genetic engineer . Studies by plant physiolo - breeding or molecular genetic engineering . gists indicate that , in a number of instances , boxylase ( RuBPCase ) . This is the enzyme re - To learn how to move genes from one organ - efficiencyof carbon dioxide fixation could be sponsiblefor fixing atmosphericcarbon diox - ism to another and have them usefully ex - 12 CALIFORNIA AGRICULTURE , AUGUST 1982 pressed , we must have model genes to work synthesized very rapidly during embryo tion of the storage proteins known as the with . The products of model genes must be globulins . ( seed ) development . Moreover , they are not recognized easily in the recipient if they are There are two major storageglobulin fami - synthesized in detectable amounts in any expressed . Soybean storage proteins seem to other tissue at any other time in the life cycle lies , the conglycininsand the glycinins . Con - be well suited to this purpose . They can be glycininsare an assemblage of three polypep - of the plant . In other words , their expression recognized by : ( 1 ) specific antibody reac - tide subunits , each believed to be one from is under strong control . Beginning with the tions ; ( 2 ) their electrophoretic mobilities on three sets of slightly different polypeptides . fertilized egg , cell division is very rapid , and ( 3 ) peptidemaps ; ( 4 ) specificamino acid gels ; a small number of virtually every cell present in the mature seed Each set is coded for by sequences . We also have recombinant DNA genes that arose by duplication from an an - is laid down . This period ends with the for - probes to detect their expression at the cestral gene during evolution . After duplica - mation of the tiny globular heart - stage em - mRNA level . tion , the genes underwent some slight bryo . Very little if any storage protein is pres - Although not quite as simple as the food changes in sequence . In fact , the three sets of ent in the embryosat this stage . Rapid growth chemistshad thought , it is stilltrue that a rel - genes coding for the three subunits probably of the embryo by cell expansion follows , ac - of storageglobulins are atively small number represent three more divergent branches , all companied by rapid synthesisand accumula - derived from one original DNA sequence . Together , they constitute what is called a multigene family . The other major storage protein , glycinin , is thought to consist of assemblages of six large and six small subunits , all encoded in a multigene family . Each gene in the genes of the family directs the synthesis of one poly - peptide that includes both a large and a small subunit arranged in tandem . These polypep - tides are subsequentlycleaved and assembled into glycinin molecules . During soybean seed development , other recognizable gene products accumulate in of these products are smaller amounts . Some known by their biological activities as pro - tease inhibitors ( the Kunitz trypsin inihibitor on the graph ) , red blood cell agglutinin , and the like . Others , such as the 15 kd protein , are known only by their electrophoreticmobility . of the storage Synthesis and accumulation proteins and the other embryo - specific pro - teins are accompanied by an increasing amount of mRNA . The amount of mRNA directing the synthesis of these proteins in the tissue can be measured by titration with cloned cDNA , that is , with DNA that has been enzymatically synthesizedusing mRNA molecules as a template , then cloned , selected , and amplified in bacteria . The of each mRNA is modulated slightly amount differently during soybean seed develop - ment ; all reach a maximum at the time of most rapid protein synthesis , and all decline as the embryo matures . Since the recombinant DNA probes will hybridize with homologous nuclear DNA from plant cells , they can be used to studythe organization of genes in the soybean genome and how it may control expression . They can also be used to detect specificDNA sequences ( such as the soybean storage protein genes ) introduced into other species in genetic en - gineering experiments . R . William Breidenbach , Anronomist , Plant Growth Laboratory , Agronomy and Range Science , U.C . , Davis ; Robert B . Goldberg , Asso - ciate Professor , Biology , U.C . , Los Angeles ; Scott E . Taylor , Post Graduate Research Plant Physiologist , Plant and Soil Biology , U.C . , Berkeley . CALIFORNIA AGRICULTURE , AUGUST 1982 13