[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Starch Synthesis in the Maize Seed

10. Starch Synthesis in the Maize Seed

L. CURTIS HANNAHPlant Molecular and Cellular Biology and Department ofHorticultural Sciences, UniversityofFlorida , P.O. Box 110690 , 2211 Fifield Hall , Gainesville , FL, USA

ABSTRACT. This chapter describes starch synthesis in seeds, with emphasis on Zea mays L.Supplementary data from other non-photosynthetic organs such as the potato tuber as well asfrom some lower organisms are reviewed in the theme of biological universality and diversity.The approach taken is genetic. The most significant insight into this pathway (as well asvirtually all others) has come from the elucidation of the biochemical lesions associated withthe many mutants, primarily in maize, that affect starch content and composition . Hence,mutants are highlighted in this review.

Introduction and Overview

Our understanding of starch biosynthesis, particularly in maize endosperm,is robust. Many mutants are known and the enzymology associated with indi-vidual mutant genes is entirely clear in many cases. Genes are expressedusually at high levels and in vitro enzyme assays are routine for virtually allsteps . Historically, the study of starch synthesis in maize has benefited fromthe abundance of mutants, their easily scoreable phenotypes, the large size ofthe maize seed, and a critical mass of early investigators of maize who wereinterested not only in the genes themselves, but also in the many interestingbiological phenomena that could be studied through their effects on thesegenes. More recent studies of starch synthesis have been stimulated not onlyby the advent of gene cloning technologies, but also by commercial interestsaimed at genetic modification of starch content and composition. This chap-ter is written not only for those interested in starch biosynthesis but also forthose involved in studies of other pathways in plants and other organisms. Thedetailed analysis of starch mutants has uncovered complexities, most notablyin biological redundancy, that will likely be found in other pathways. Hope-fully, the vast amount of effort needed to decipher these complex phenomenain starch synthesis will aid in subsequent studies of other pathways.While mutants have been invaluable in deciphering the starch biosynthetic

pathway, the same mutants have also been instrumental in studies of otheraspects of biology. Most notable is the fact that Mendel's wrinkled pea geneaffects starch biosynthesis. Hence the whole discipline of genetics began

B.A. Larkins and IX. Vasil (eds.), Cellular and Molecular Rio/OKY ofPlant Seed Development. 375-40 5.© 1997 Kluwcr Academic Publishers.

376 L. Curtis Hannah

with a starch mutant! Of perhaps comparable importance was the discoveryof transposable elements. Here too starch mutants played a pivotal role . Theclose linkage and endosperm expression pattern of three genes, including thestarch synthetic genes shrunken] and waxy, allowed McClintock to monitorthe movement, and therefore the existence, of the first-described transposableelement, Dissociation.Modem studies of starch synthetic genes benefited directly from investiga-

tions aimed at the study of plant transposable elements. With the developmentof gene cloning and sequencing technologies, a molecular description of trans-posable elements became a feasible goal. Viewed as traps or resting sites fortransposable elements, wild type starch synthetic genes were first cloned .These were then used as probes to isolate interesting mutant alleles contain-ing the various transposons. Hence, the early investigations concerning thestructures of the various starch synthetic genes came as spinoffs from studiesaimed at a totally different goal, the molecular description of transposableelements.Genetic dissection of the starch biosynthetic pathway is emphasized in this

chapter. Detailed descriptions are given in those cases in which the prima-ry function of a gene is known and loss-of-function mutants are available .Genes are described in terms of their mutant phenotypes and their primarylesions in the enzymology of starch synthesis. This information is then usedto gain understanding into the pathway of starch synthesis. Interestingly, pre-cursor/product relationships that one might surmise from the structures of thevarious glucose polymers do not always hold up to genetic analysis.The term starch refers to a series of polymers of glucose held together

through alpha 1,4 bonds. The simplest of these is amylose. This straight chainpolymer accounts for approximately 30%of the starch in a normal maize seed.While amylose is considered a straight chain molecule, some polymers of thisclass contain occasional branch points due to alpha 1,6 linkages. The majorityof the glucose , accounting for virtually all of the remaining starch, is foundin a more complex molecule, amylopectin. Amylopectin resembles amyloseexcept for its higher frequency of branches involving alpha 1,6 linkagesbetween the glucose residues . These branches account for 4-5% of the glucoselinkages in amylopectin. A more highly branched polymer, phytoglycogen,contains approximately 10% alpha 1,6 linkages. Operationally defined as awater soluble polysaccharide (WSP), phytoglycogen is extremely abundantin maize seed lacking a functional sugary] (Su) allele. The interesting genesisof phytoglycogen is discussed in detail below. A detailed description of thevarious components of starch can be found in the recent reviews of Nelsonand Pan (1995) and Martin and Smith (1995).A listing of the key enzymes for which mutant, molecular, biochemi -

cal and, in some cases , enzymological data are available is given in Table I.

Starch Synthesis in the Maize Seed 377

Sucrose

(1*Glucose + Fructose, ,

'. "Sucrose

(2)t

UDP-Glucose + Fructose

(3)1 ¥~f jF (4)

Glucose-1-Phosphate +ATP~ ADP-Glucose + PP~__ (5)

ADP-Glucose + (Glucose)n -.. Amylose + ADPt (6)

Amylopectin Precursor(7)t .Amylopectin(7)t .(8)

Phytoglycogen

Fig . 1. Pathway for starch biosynthesis. This pathway is based exclusively on the analysis ofmaize mutants affecting starch synthesis in the maize endosperm . Steps for which mutants areavailable are assigned a number. Specified enzymes are listed in Table I and in the text.

A possible starch biosynthetic pathway, based on genetic data , is shown inFigure 1. While there exist substantial genetic data to support this pathway,it is currently unclear whether all of starch synthesis occurs by this pathway.For example, starch phosphorylase, which is capable of synthesizing starch invitro without a sugar nucleotide substrate, peaks during the most active periodof starch synthesis in the developing endosperm. The appropriate mutants toevaluate phosphorylase involvement in starch synthesis are not in hand.

The Pathway

In the vast majority ofplants, and certainly in the case ofthe maize endosperm,the form of sugar transported from the leaf to the sink tissue is sucrose. Thusthis review will begin with this sugar and end with polysaccharides.


TABLE I

Gene-enzyme relationships in starch biosynthesis of the maize endosperm. Gene symbolsare defined in the text.

Step Enzyme Gene Comments

Invertase Mnl Relationship between gene and

enzyme awaits elucidation2 Sucrose synthase Shl Both genes have been

Susl cloned and sequenced

3 UDP-glucose pyrophosphorylase Anti-sense variant in potato4 ADP-glucose pyrophosphorylase Sh2 Both genes have been

Bt2 cloned and sequenced

5 Membrane-bound Btl Gene has been clonedmetabolite transporter and sequenced

6 Granule bound ADP-glucose Wx Gene has been cloned

glucose transferase and sequenced

7 Starch branching enzyme Ae Gene has been cloned

and sequenced8 Starch debranching enzyme SuI Gene has been cloned

and sequenced

Sucrose catabolism

Accumulating data strongly point to sucrose appearing twice in the path tostarch synthesis. Original synthesis of sucrose occurs in green tissue andits resynthesis occurs after entry into the kernel. This surprising inference,originally based on pulse-chase experiments with 14C02, is supported bymore recent genetic data, as well as feeding experiments with sugars otherthan sucrose.The original observation leading to the realization of a possible sucrose

degradation and resynthesis pathway in the maize kernel was made by Shan-non (1968) who fed 14C02 to maize leaves and then followed the label throughvarious sugars in the seed at different periods after labelling.The first sugar labelled was sucrose, followed in time by reducing sugars.

Surprisingly, the label then was again found in sucrose. Labelled sucrose wasshown to be transported to the kernels, but it was cleaved in the pedicel priorto transfer into the endosperm. The labelled monosaccharides, glucose andfructose apparently were then converted back to sucrose prior to its utilizationin starch biosynthesis.If maize kernels can synthesize sucrose, it may be possible to feed develop-

ing kernels reducing sugars and synthesize sucrose. This hypothesis was tested


and a positive result was obtained by Cobb and Hannah (1986) who used anin vitro kernel development protocol (Gengenbach 1977) to feed developingkernel s on a med ium containing glucose or fructose. The original mediumdescribed by Gengenbach contains sucrose as the carbon source. Cobb andHannah found that kernels develop on reducing sugars and, more importantly,showed that kernels grown on reducing sugars synthesized sucrose, a predic-tion made earlier by the 14COZ feeding experiments.The enzyme synthesizingsucrose is not the predominant endosperm sucrose synthase (Cobb and Han-nah 1988) and it is reasonable to suspect that sucrose phosphate synthase isthe relevant enzyme.While mutants have not yet been described for the hypothesized sucrose-

synthesizing enzyme, there do exist genetic data invoking the importanceof two sucrose catabolizing enzymes, sucrose synthase and invertase. Theimportance ofsucrose synthase, as based on genetic/molecular/enzymologicaldata , is inescapable. While this cannot yet be said for invertase, a firm casefor its importance is building. Current data suggest that invertase functionsin the base of the seed to cleave the entering sucrose (reviewed below). Afterresynthe sis of sucrose, sucrose synthase leads to the cleavage of sucrose andthe synthes is of fructo se and UDP-glucose. One or both of these then serveas substrates for starch synthesis.

Step 1. Invertases and their genetic control

Characteri zation of the mutant miniature (Mn1) may aid in the elucidation ofthe role of invertase in starch synthesis. The mn1 mutant was described 50years ago (Lowe and Nelson 1946). As the name implies, seeds homozygousfor this mutation are smaller than normal. They also contain less solublesugars and a greater proportion of sucrose relative to their normal counterparts(reviewed in Nelson and Pan 1995 ).Mill er and Chourey (1992) report ed thatmn1 mutants have greatly reduced

invertase within the developing seed and in the underlying maternal tissue.Mutants ofmn1 behave as typical Mendelian mutations and therefore reduc-tion of maternal inverta se was surprising. Furthermore, since it is knownthat the various invertases (soluble, cell wall bound , alkaline and neutral) areencoded by separate structural genes in maize, (Xu et aI., 1995; K. Koch, per.com. ), the mechanism by which mutation at a single gene reduce s all theseenzymic activities must not be simple.Is the reduction in invertase activity the cause or the consequence of the

reduced seed size and other pleiotropic effects associated with the mnImutant? For example, the MnI gene product might be associated with thedevelopment or differentiation of basal endosperm cells. Their failure todevelop might give rise to a whole host of symptoms in this region of thedeveloping seed, including the loss of all the invertases. Recent work fromChourey and colleagues (Taliercio et aI., 1995) suggests that Mn1 is in fact


a structural gene for one of the invertases. They generated a series of EMSinduced mnl mutants and probed these with a cell wall invertase cDNAclone . While some of the mutants lacked a transcript (thereby being non-informative), others exhibited a detectable product. One mutant containedwild type levels of a transcript indistinguishable in size from wild type and anextremely leaky phenotype. When homozygous, the mutant conditions a wildtype phenotype and can only be identified as mutant when heterozygous withmore severe mnl mutant alleles. However, the mutant is reported to be quitelow in invertase activity, thereby complicating interpretation. Perhaps, themutant enzyme is particularly labile following extraction. This would explainthe unexpected, low levels of invertase seen in vitro.Currently, the role of Mnl in kernel development is not fully understood.

Perhaps future data will definitively show that it is a structural gene encodingthe cell wall-bound invertase. The gene represents an excellent candidatefor transposon tagging, since this approach requires no prior biochemicalknowledge of the gene's product.Analysis of additional starch mutants provides some evidence that sucrose

cleavage and resynthesis provide a gradient important in the flow of sugarsfrom source to sink tissue, as has been argued from conventional physiologicalstudies . Were starch synthesis per se the driving force for the flow of sugarsfrom leaves to developing kernels, then one would not expect elevated sugarlevels in mutants markedly defective in the latter stages of starch synthesis.Severe mutants such as btl (brittle]), bt2, and sh2 accumulate up to 20-timesnormal levels of sucrose and in some cases sucrose accounts for up to 50%of the dry weight in these mutant seeds.

Step 2. Sucrose syntheses and their genetic control

The major form of maize endosperm sucrose synthase is encoded by theshrunken] (Shl) locus on chromosome 9. This gene, one of the first describedand used by early maize geneticists, was defined by mutants which developa cavity within the endosperm and a shrunken kernel. The phenotype issometimes classified as mild, at least in comparison to severe loss-of-functionmutations at other loci such as sh2, btl and bt2.The first biochemical investigations of Shl were done by Schwartz (1960)

who showed that an abundant, buffer-soluble, endosperm protein was missingin a series of independently-isolated shl mutants. Subsequently, work fromOliver Nelson's laboratory showed that the biochemical lesion associatedwith shl mutants is sucrose synthase (Chourey and Nelson 1976, 1979). Theenzyme catalyzes the synthesis of sucrose and UDP from UDP-glucose andfructose. ADP and ADP-glucose can also serve as substrates, although mostisoforms prefer the uracil containing substrates.What is the physiological role of sucrose synthase in the maize endosperm?

Data reviewed above showed that the maize seed has the ability to cleave as


well as to synthesize sucrose. Two conceptually different approaches havebeen taken to address the function of sucrose synthase and both led to thesame conclusion . Chourey and Nelson (1979) took advantage ofa genetic phe-nomenon that occurs rarely, especially in plants: intracistronic or interalleliccomplementation. Whil e the phenotype resulting from the cross of two reces-sive mutants is routinely used to classify the mutants as allelic or nonallelic ,in rare instances , allelic mutants sometimes exhibit a wild type phenotype.The conclusion that the mutants are indeed allelic can only be drawn in casesin which a relatively large series of mutant alleles is examined, and eachof the mutant alleles in the complementing heterozygote typically producesmutant phenotypes in crosses with each of the other mutants. This criterionwas fulfilled in the case of the shl mutants.Analysis of sucrose synthase in the compl ementing shl mutants showed

that while sucrose synthesis activity was the average of the two mutant alleles,sucrose degradation activity was elevated, relative to the two parents, inthe complementing heterozygote. Since the change in the enzymologicalphenotype in the direction of sucrose cleavage, but not sucrose synthesis, wasassoc iated with the change in kernel phenotype, the authors concluded thatthe physiological role of sucrose synthase is actually sucrose degradation.These experiments are noteworthy for a number of reasons. First, these

studies provided the first evidence that sucrose synthase is actually sucrosedegradase, a conclusion that has held up in subsequent experiments. Second ,the data bear on the concept of threshold amounts ofenzyme needed for a wildtype phenotype. The complementing heterozygote conditioned 10%wild typesucrose synthase activity, whereas only 7% of wild type enzyme was foundin each of the single mutants. Clearly, these data show that much less than100% - in this case only 10% - of the wild type enzyme amount is neededfor the wild type phenotype. Finally, the data raise a number of questionsconcerning enzymology and the mechanism of action of sucrose synthase. Apriori, one would expec t that the same sites are used for binding the substratesand products of the reaction, and hence it is difficult to reconcil e elevation inactivit y in only one of the two reversible directions. At least superficially, thismay represent a violation of the Haldane relation ship in which the equilibriumof a reaction can be expressed in terms of the Kms and Vms of the forwardand backward reactions.The role of sucrose synthase was addressed independently by Cobb and

Hannah (1986, 1988) . Since wild type seed can metabolize reducing sugarsin the growth medium, produce sucrose, and synthesize a normal-appearingviable seed, these investigators asked if seeds containing the shl lesion alsohad this ability. If sucrose synthase is important in sucrose synthesis, then onewould expect that the ability to synthes ize sucrose from reducing sugars inthe growth medium would be impaired . This was not observed. Pulse-chaseexperiments showed that the rate of conversion of labelled reducing sugars to


sucrose was not diminished in shl developing seed, providing further supportfor the idea that sucrose synthase actually functions to degrade sucrose.If sucrose degradation by sucrose synthase is important in starch synthesis,

then one or both of its product s, UDP-glucose and fructose, must be keymetabolite(s) for starch synthesis. UDP-glucose is important in cell wallsynthesis and therefore , as first sugges ted by Cobb and Hannah (1983 ), onemight reasonably classify shl as a cell wall mutant as well. In fact, BarbaraMcClintock (per com.) came to this conclusion many yea rs before any of thebiochemi stry of Shl was understood. She monitored sectors of cell s exhibitingthe wild typeWx function in a background of mutant cells. Individual clones ofcells could be traced back to a single transposition event restoringWx function.In a wild type background, the mosaic pattern could be traced to a point atthe center of the endosp erm. However, when this pattern was studied in a shlbackground, the pattern did not terminate at a single point, but rather in a massof tissue. Her interpretation was that the integrity of cell s was destroyed in ashl background, but not in wild type. She also realized that the severity of theshl phenotype was variable through the endosperm. These interpretations andconclu sions of McClintock are consistent with recent studies from Starlingerand Chourey (reviewed in Chourey and Taliercio, 1994) showing localizationof the various sucrose syntheses in the endosperm.These studies demonstratedthat expression of the Shl-encoded isoform of sucrose synthase (as well as thesecond sucrose synthase encoded by Sus described below) varies in differentcells of the endosperm.Loss of Shl function does not condition the severely shrunken pheno-

type seen with some other starch mutants. A priori , three explanations couldaccount for this: ( I) all shl mutants examined to date are leaky in the sensethat the mutant alleles condition a low but physiologically significant levelof enymic activity, (2) a second sucrose synthase exists that provides fruc-tose and/or UDP-glucose allowing for the residual starch synthesis, or (3) aparallel pathway exists for the conversion of sucrose to starch which doesnot involve sucrose synthase. Despite significant effort, this question remainsunanswered. However, some progress has been made as described below.Following the cloning of the Shl locus, Burr and Burr (1981) showed that

much of the shl allele of the McClintock shl bzlm4 stock had been deleted inthe derivation of a complex gene rearrangement. Chourey et al. (1986) foundthat this stock contained detectable amounts of sucro se synthase. Hence, thecombination of molecular and enzymological analysis demonstrated a secondsucrose synthase gene expressed in the maize endosperm. Thi s second sucrosesynthase locus, now termed sucrose synthase! or Susl , was cloned (McCartyet aI., 1986) and its structure was recentl y publi shed (Straw et aI., 1994).While the existence of this duplicate sucrose synthase locus encoding a lowlevel of enzymic activity readily provides an explanation for the relativelyhigh levels of starch found in the shl mutants, Chourey and Taliercio (1994)presented data which they interpret to show that loss of Susl function gives


rise to no discernible phenotypic change in the maize plant. Furthermore,plants doubly mutant for Susl and Shl are reported to be no more severe inphenotype than that conditioned by the loss of just Shl function. These data,if confirmed, argue that another enzyme, perhaps invertase, is responsible forthe residual starch found in shl mutants.The interpretation of Chourey and Taliercio (1994) suggests that the Susl

gene is dispensable. Shaw et al. (1994) re-examined this conclusion. Theysequenced the Susl gene and compared it to Shl. The genes were nearlyidentical except for one intron found only in Shl. Fourteen of the 15introns arein identical positions in the two genes. Importantly, while the exon sequencesexhibited significant similarity, no such pattern was found in intron sequences .The facts that the genes encode different isoforms of the same enzyme, exhibitsequence similarity in coding regions, and have virtually all introns in identicalpositions argue that the genes share a common origin. They also argue thatthe introns (at least 14 of the 15) were present before the duplication. Ifso, the introns and exons diverged at radically different rates. Barring someunprecedented mechanism of differential rates of mutation in exons versusintrons, one is forced to conclude that selection played a major role in thedifference in the extent of sequence identity of exons and introns . Our currentunderstanding of introns suggests that only sequences at exon/intron borderssignify functional introns, hence, it appears that much sequence diversitywithin introns can be tolerated in functional introns. On average, mutationsin introns rather than exons are much more likely to be tolerated in functionalgenes.The data reviewed above strongly suggest that selection pressure played a

significant role in the evolution of Shl and Sus}, a conclusion not expected ifSusl lacks an important physiological function. Furthermore, every monocotexamined in sufficient detail contains a Sus- and a Shl- type gene [termedSus2 when referring to all plants (Hannah et al., 1994)] of sucrose synthase(reviewed in Shaw et aI., 1994), a conclusion unexpected if Susl is dispens-able. Current data do not allow us to formally exclude the possibility thatSusl played an important role in the physiology of a progenitor ofmaize (andperhaps all plants), whereas modern maize (and perhaps all plants) does notrequire an active Susl gene. This hypothesis would explain the vast differ-ences in the rates of divergence exhibited by exons and introns and wouldexplain the lack of phenotype of the maize susl mutant. Perhaps invertase hastaken over the role of the Sus} encoded sucrose synthase in progenitor plants.Alternatively, perhaps there is a third sucrose synthase yet undetected in maizeendosperm. Further insight into the present susl mutant may be needed toresolve this important question. The possibility that growth conditions existwhich distinguish Susl and sus} plants/tissues has yet to be analyzed in detail.


Step 3. UDP-glucose pyrophosphorylase and anti-sense mutants

Evidence was reviewed above that sucrose synthase functioning as sucrosedegradase is an important enzyme in maize endo sperm cells actively synthe-sizing starch. Sucrose synthase catalyzes the conversion of sucrose and UDPto UDP-glucose and fructose. Conventional schemes of starch (and cell wall)synthesis then invoke a role for UDP-glucose pyrophosphorylase (UGP).This is an extremely active enzyme in the maize endosperm (20-times that ofADP-glucose pyrophosphorylase) that converts UDP-glucose and pyrophos-phate to UTP and glucose-l-phosphate. The latter product is then used byADP-glucose pyrophosphorylase for the synthesis of ADP-glucose, the actu-al substrate for starch synthesis .Despite the importance of UGP, plant mutantsaffecting it have not been found. Moreover, Zrenner et al. (1993) produced aconstitutive anti-sense version of this gene and placed it into potato. Whileendogenous enzyme levels were decreased 95 to 96%, there was no detectablechange in starch synthesis or plant growth and development. This unexpectedresult has two explanations. Either a very small amount of wild type enzymelevel is sufficient for normal starch synthesis or the enzyme is unimportant.

Step 4. ADP-glucose pyrophosphorylases and their genetic control

The next step in the flow of sugars to starch for which mutants are availableis the synthesis of ADP-glucose and pyrophosphate from glucose-l-P04and ATP by ADP-glucose pyrophosphorylase (AGP). The general, if notuniversal, importance of this enzyme in polysaccharide biosynthesis wasmade evident by the discovery of mutants in several plants, as well as bacteria(reviewed in Nelson and Pan 1995), that greatly reduce activity of AGPand polysaccharide synthesis. The genes shrunken2 (Sh2) and brittle2 (8t2)encode the two subunits of the maize endosperm tetrameric AGP (Hannahand Nelson 1976). Both genes have been cloned and sequenced (Bhave etal., 1990, Bae et al., 1990, Shaw and Hannah 1992). Bt2 encodes the smallsubunit , whereas Sh2 encodes the large subunit of AGP.That maize endosperm AGP is composed of two different subunits (Hannah

and Nelson 1976) was unexpected (and unaccepted by some investigators),since it was known that E. coli AGP was a homotetramer (reviewed in Preissand Levi 1980). However, the clon ing of Sh2 and Bt2 (Bhave et al., 1990; Baeet al., 1990) and the AGP structural genes from other plants (for example ,Okita et al., 1990) provided definitive evidence for the existence of twodissimilar subunits in plants (reviewed in Smith-White and Preis s, 1992).While Sh2 and Bt2 are complementary genes, they likely share a common

origin. Significant sequence similarity exists between the Sh2 amino acidsequence and the subunit of the homotetrameric E. coli AGP encoded byglg-C (Bhave et al., 1990). Sequence similarity is also apparent between theBt2 and glg-C proteins (Bae et al., 1990). A relationship between the large

Starch Synth esis in the Maize Seed 385

and small AGP subunits is seen with other plants as well (Smith-White andPreiss, 1992). These facts point to an intriguing evolution of Sh2 and Bt2.These genes (as well as their counterparts expressed in other tissues and inother plants) most likely arose from an initial duplication event. Followingthis, independent mutations with in Sh2 and Bt2 separated them such that theSH2 and BT2 proteins were no longer interchangeable. Yet the protein s couldstill interact to form an active enzyme. A more detailed discussion of therelationship between Sh2 and Bt2 can be found elsewhere (Hannah et aI.,1993; Boyer and Hannah 1994). The nature of the selection pressure leadingto the formation of complementary genes from initially duplicate loci is anintriguing unresolved issue.Other duplications of the AGP structural genes have also occurred, as

revealed from studies of tissue- specific expression patterns. As is the case withother maize endosperm starch synthetic enzymes, different tissues expressdifferent sets of structural genes for AGP. Early enzymological and geneticstudies showed that the endosperm and embryo contain different AGPs (Preisset aI., 1971, Hannah and Nelson , 1976). Enymological studies showed thatthe pollen (Bryce and Nelson, 1979 ) and the leaf (Fuchs, 1977) AGPs are notreduced in sh2 and bt2 mutants. Subsequent molecular investigations haveconfirmed the tissue-specific nature of AGP expression and have uncovered anumber of other interesting attributes. Giroux and Hannah (1994) cloned theembryo transcripts hybr idizing to Sh2 and Bt2 clones. Hybridization of RNAwith Sh2 and Bt2 clones (Giroux and Hannah , 1994), genetic mapping (Burret aI., 1991), and sequence analysis (Giroux et aI., 1995) all showed that thegenes expressed in the embryo are not Sh2 and Bt2. Furthermore, the Bt2counterpart in leaf tissue (Prioul et aI., 1994) was isolated and shown fromsequence analysis not to be Bt2 or the embryo small subunit encoded by theAgp2 gene.Like the situation described for endosperm sucrose syntheses , a second set

of AGP struc tural genes is expressed in the endosperm, albeit at low levels.Definitive proof for this activity was provided by sh2 and ht2 mutants shownto be null at the protein or RNA level , but which still retained detectable AGPlevels (Giroux and Hannah, 1994). Furthermore, transcripts could be foundin mutant endosperms that show strong hybridization to the maize embryoAGP structural genes,AGP] and AGP2.Thus, it appears that like the case forShl and Sus] , Agpl and Agp2 may be expressed in the endosperm as well asin the embryo.Whil e it is dear that a series of genes encodes the small and the large

subunits of AGP, sequence analysis shows that the rate of divergence withinthe small subunit gene family is much slower than that of the large subunit.For example, a compar ison of the Bt2 sequence with that of L2, the maizegene encoding the leaf sma ll subunit, revealed 96% identity throughout mostbut not all of the genes . In contrast, the genes encoding the large subunitshave diverged to the point that the Sh2 probe cannot detect the leaf large


subunit. An inspection of large and small subunit sequences compiled bySmith-White and Preiss (1992) shows that the differential rate of divergenceof the two subunits occurred in other plants as well. These data have atleast two interpretations: (1) one could argue that the small subunit is moreimportant to enzyme activity and the changes in the large subunit representneutral mutations or (2) the changes within the large subunit are importantand the differences distinguishing the various AGPs evolved in response todifferent physiological conditions.

AGP represents an important rate-limiting step in starch biosynthesis

Without argument, AGP has received the most attention from biochemistsinterested in studying rate limiting steps in starch biosynthesis. ADP-glucoseis the first unique intermediate in the bacterial glycogen and starch syntheticpathway and AGP is an allosteric enzyme (reviewed in Preiss, 1982; Preiss andLevi, 1980; Preiss et al., 1991). In almost every organism and tissue analyzed,AGP has proven to be an allosteric enzyme. In bacteria, various moleculesactivate AGP, with the most effective one arising from the preferred carbonsource. An E. coli mutation termed glgC-16 has altered allosteric propertiesand also synthesizes elevated amounts of glycogen.Plant AGPs are activated by 3-phosphoglyceric acid (3-PGA) and inhibited

by phosphate, however, genetic data bearing on the rate-limiting role forthis enzyme in higher organisms were not available until recently. Stark etal. (1992) cloned the E. coli glgC-16 mutant and expressed it in tubers oftransgenic potato plants and increased starch content 30%. These resultsclearly show that AGP represents a key regulatory step for starch synthesisat least in potato tubers. Giroux et al. (1996) used the transposable element,Dissociation (Ds), to create genetic variation within a region of the maizeSh2 gene thought to be important in the allosteric properties of AGP. Anexcision event of Ds (Rev6), which left a six base pair insertion at the site ofDs integration, led to a 15 - 18% gain in seed weight. The variant does notincrease the percentage of starch and thus the alteration in starch biosynthesisapparently increases the synthesis of other endosperm components. In effect,the alteration in starch synthesis creates a stronger sink for the flow of carboninto the maize seed.Characterization of the mutant AGP suggests the six base pair insertion

leads to phosphate insensitivity. This was found for the enzyme extractedfrom the developing endosperms, as well as in wild type and Rev6 AGPexpres sed in E. coli (Giroux et al., 1996). The six base pair insertion is likelyuniversally important since its insertion into the potato tuber large subunitalso led to a phosphate insensitive AGP.While the Rev6 variant of Sh2 provides genetic evidence for the importance

of phosphate inhibition ofAGP, no alteration in 3-PGA activation was noted.3-PGA activation of AGP was first noted in chloroplasts. Here activation by


the triose sugar has a ready explanation, since its buildup during photosynthe-sis would trigger starch synthesis and hence a mechanism to stockpile sugarsfor later transport to sink tissues. While this provides a logical explanation for3-PGA activation of AGP in the chloroplasts, it does not explain the activationof the enzyme in non-photosynthetic tissue.There is no good explanation for 3-PGA activation of endosperm AGP,and

it is also relevant that endosperm AGPs do not show the extent of activationexhibited by the chloroplast enzymes. Hannah and Nelson (1975) and Dickin-son and Preiss (1969) reported that maize endosperm AGP shows less 3-PGAactivation than the chloroplast enzyme. This led Hannah and Nelson (1975)to speculate that this activation may not be physiologically relevant. If thegenes encoding the endosperm AGPs were derived through evolution fromthose encoding the chloroplast activity, then the activation properties maysimply represent evolutionary baggage. Indeed, the recent report that barleyendosperm AGP is not activated by 3-PGA (Kleczkowski et al., 1993) sug-gests that 3-PGA activation of AGP is not a prerequisite for starch synthesisin the endosperm.That the reported low levels of 3-PGA activation of endosperm AGPs

reflect the situation in vivo was questioned by Plaxton and Preiss (1987).They noted that the small subunit (BT2) undergoes proteolytic cleavage,giving rise to a protein approximately 1 kDa smaller than the intact protein.They showed that the purification procedure of Dickinson and Preiss (1969)led to proteolytic cleavage of the BT2 protein, and they also showed thatthe extent of 3-PGA activation of their enzyme preparation was higher thanthat reported in the earlier studies of Dickinson and Preiss (1969). Plaxtonand Preiss (1987) concluded that cleavage of the BT2 protein decreased 3-PGA sensitivity. In subsequent studies, Hannah et al. (1995) showed thatnot only the BT2 protein, but also the SH2 protein undergoes proteolyticcleavage. These workers could not detect differences in 3-PGA activationdue to proteolytic cleavage, but did note that different maize lines exhibitvarying degrees of activation and point out that the differences between theresults of Plaxton and Preiss and Dickinson and Preiss could be due to thedifferent maize lines used in the separate experiments. At this juncture , itis still unclear whether 3-PGA activation of non-photosynthetic AGPs isphysiologically important and whether the properties of extracted enzymemimic the in vivo situation. Expression of AGP genes from different tissuesand from different plants in a common genetic and physiological background(e.g . E. coli) may resolve this important question.

Step 5. The brittle (Btl) gene and its protein

The Btl locus on chromosome 5 was the first of the maize starch syntheticgenes cloned by transpo son tagging (Sullivan et al., 1991). Sequence analysisand data base comparison showed the BTl protein is related to a family


of membrane-bound, metabolite transporters. Greatest similarity was foundwith an adenylate translocator. As predicted, antibody synthesized againstthe BTl protein reacted with a membrane-bound protein surrounding thestarch granules (Sullivan and Kaneko, 1995), and with membranes isolatedfrom maize endosperm amyloplasts (Cao et aI., 1995). Furthermore, the BTlprotein can be imported into pea chloroplasts (Li et at., 1992).

The metabolite transported by the BTl protein

Studies of the btl mutant clearly show that the BTl protein is important forstarch biosynthesis and therefore the elucidation of the molecule transportedby this protein is imperative for a complete understanding of starch synthesisin the maize endosperm. Conventional wisdom suggests that AGP is locatedwithin the amyloplasts (reviewed in Martin and Smith, 1995) and thereforethe transported molecule should precede the synthesis of ADP-glucose. Starket aI. (1992) showed that a transit sequence on the E. coli AGP was necessaryfor activity in potato, providing strong genetic evidence for an amyloplasticlocalization of AGP.Is the exclusive localization ofAGP in amyloplasts, as noted in potato, true

for all plants? Emerging data strongly point to a negative answer. Perhaps thestrongest evidence favoring a cytoplasmic location ofAGP comes from studiesof the barley endosperm. Work appearing in abstract or review form (Villandand Kleczkowski , 1994; Kleczkowski et aI., 1995) shows that antibody madeagainst E. coli- expressed barley AGP reacts primarily with proteins localizedin the cytoplasm. Similar results have also been reported for tomato fruit AGP(Chen and Janes, 1995).If AGP is located in the cytoplasm, then ADP-glucose could be the sugar

moiety translocated by the BT1 protein. Substantial uptake of ADP-g1ucoseinto amyloplasts for starch synthesis has been noted in a number of studieswith diverse plant tissues (for example, Pozueto-Romero et aI., 1991a, b, Tet-low et aI., 1994), these results were interpreted as being either physiologicallyirrelevant or proof of the unimportance of AGP. Obviously these conclusionsmust be reevaluated.Shannon and colleagues (Liu et aI., 1992, Shannon, per. com.) showed

that amyloplasts from developing maize seed can take up ADP-glucose andincorporate the sugar into starch. Most importantly, this activity is greatlyreduced in btl mutants. These observations allowed Shannon and coworkersto test an hypothesis concerning the location of AGP. If AGP is exclusivelylocalized within the amyloplasts and the BTl protein transports a sugar pre-ceding AGP in the starch biosynthetic pathway, one would expect the amountof ADP-glucose to be greatly reduced in a btl mutant. However, the oppositeresult is true. Levels of ADP-glucose are 13-fold higher in btl endosperms.Perhaps this arises from an enzymic activity independent ofAGP (e.g. sucrosesynthase or the minor AGP); however, this elevation does not occur in btl


sh2 double mutants This provides definitive evidence for the importance ofthe major endosperm AGP in conditioning the elevated ADP-glucose in btlmutant endosperms. These data clearly point to a cytoplasmic location of thethe major endosperm AGP.Conversely, there is some qualitative evidence for an amyloplastic location

of maize endosperm AGP.Cao et al. (1995) isolated amyloplasts from normalendosperms and found SH2 and BT2 proteins were present in the stroma, butnot in the amyloplast membranes. Miller and Chourey (1995) report, fromimmunolocalization investigations that antibodies against SH2 and BT2 crossreact with proteins located on the amyloplasts . Their photographs also showabundant cross reactivity in the cell wall and in the cytoplasm. While there isevidence for at least some amyloplast localization of AGP, neither the studiesof Cao et al. (1995) nor Miller and Chourey (1995) allow for a quantitativeestimation of the total amount of AGP in the amyloplasts.AGP may likely be present in both the amyloplasts and in the cytoplasm.

Two forms of AGP (Sh2 end Bt2 dependent and independent) are present inmaize endosperm. The metabolite studies of Shannon and colleagues men-tioned above provide definitive evidence for a cytoplasmic localization of theSh2/Bt2 AGP. Whether the amyloplast AGP is the minor, Sh2/Bt2- indepen-dent activity awaits further investigation. However, Shaw, Barry, and Hannah(unpubli shed) have found that the SH2/BT2 AGP,when expressed in E. coli,is active when the subunits are encoded by the complete coding regions ofboth genes. These data rule out the possibility that leader peptides must beremoved before an active enzyme is formed. These investigators also foundthat N-terminal truncations of both subunits can be made, and at least someenzyme activity is maintained. Clearly then, it is possible that the majorSh2/Bt2 encoded AGP is present in both the amyloplast and cytoplasm. Inthis regard, it is relevant that Giroux and Hannah (1994) noted that the sizesof the SH2 and BT2 proteins isolated under denaturing conditions from themaize endosperm are identical to those of the proteins expressed from fulllength clones in an in vitro transcription/translation system. Smaller proteinsare not recovered from the endosperm if care is used to abolish proteolyticactivity. If insertion into the amyloplast requires cleavage ofa leader sequence,as found in many studies of protein uptake into plastics, the data of Giroux andHannah would argue that the vast majority of maize endosperm ADPglucosepyrophosphorylase is found in the cytoplasm.

Step 6. Starch syntheses and their genetic control

While it is abundantly clear that ADP-glucose is the major if not sole sourceof glucose used for starch synthesis and that the majority of this substrate isproduced by the Sh2/Bt2 AGP, a whole host of starch syntheses use ADP-glucose for starch synthesis. These enzymes extend the amylose/amylopectinbackbone by formation of alpha 1,4 bonds between the neighboring glucose


residues. Classically, distinctions between starch syntheses are made alongtwo lines: whether the enzymic activity is involved in amylose or amylopectinsynthesis and whether the enzyme is strongly bound to the starch granule.The waxy (wx) mutations of maize provided important insight into the

complexity of starch syntheses and the pathway leading to amylose andamylopectin biosynthesis. While one would expect that amylopectin, con-taining the alpha 1,6 branch points, would be synthesized from the simpler,straight-chained amylose, analysis of maize wx mutants and subsequentlysimilar mutants in other plants, strongly suggests that this does not occur.Wx encodes a starch granule-bound ADP-glucose glucosyl transferase (heretermed simply starch synthase) (Nelson and Rines, 1962). Lack of Wx func-tion leads to a total loss of amylose but has little to no effect on amylopectincontent or composition. Thus, amylopectin apparently is not synthesized fromamylose.The elucidation of the Wx-granule bound starch synthase relationship was

accomplished through the use of simple but elegant genetic analysis. Fol-lowing the surprising report that wx mutants lacked granule-bound starchsynthase activity, the question arose whether this was a cause or consequenceof abolished amylose synthesis. As reviewed earlier (Hannah et al., 1993),the fact that starch synthase activity increased linearly with the number offunctional Wx alleles in the maize endosperm, whereas amylose content wasnearly restored with only one functional gene, ruled out the possibility thatwx starch granules lacked enzymic activity because they lacked amylose. Theindependent cloning ofWx in the laboratories of Heinz Saedler and Nina Fedo-roff, the subsequent molecular analysis of wx mutants (Echt and Schwartz,1981), and the solubilization and enzymic characterization of theWX protein(Macdonald and Preiss, 1983) provided definitive molecular evidence that Wxencodes the major starch granule-bound starch synthase.The importance of the WX protein to amylose synthesis is likely universal

in all plants .As reviewed in Nelson and Pan (1995), wx mutants in barley, rice,and sorghum also appear to be deficient in granule bound starch synthase.Potato genes, isolated by homology to the maize wx gene, inhibit amylosesynthesis when expressed in the anti-sense direction (Visser et al., 1991).Thus, the WX protein is responsible for amylose synthesis in plant systemsas diverse as maize endosperm and potato tuber.The universal importance of the WX protein for plant amylose synthesis

in plants has been challenged by A. Smith and colleagues (Smith, 1990).This group solubilized proteins from pea amyloplasts and made antibodiesto the various size-separated proteins. A protein of 77-kDa was reported tocontain starch synthase activity, whereas the WX protein in all other plants isapproximately 59-kDa. While a relatively abundant 59-kDa protein was cross-reactive to WX antibodies, it had very little starch synthase activity. Sivak etal. (1993) solubilized pea amyloplast proteins and, in contrast to the work ofSmith, found the majority of starch synthase activity associated with the 59-


kDa protein. In response to this report, Martin and Smith (1995) proposed thatproteolytic cleavage of the larger, 77-kDa authentic starch synthase gave riseto an enzymically active degradation product, fortuitously the same size as theWX protein. While this possibility seems unlikely, additional investigationappears necessary to resolve this point. Analysi s of amylose-free mutants ofpea would clearly be relevant.The analysis of wx mutants clearly pointed to the existence of isoforms of

starch synthase, and indeed such forms have been found. Two soluble formsof starch synthase (SSS) were reported from the maize endo sperm (Ozbun etaI., 1971). Not only can they be distinguished by behavior during purification,but they also differ in substrate specificity and the requirement for an addedprimer.Recent evidence points to the possibility that one of the SSS isoforms

may also be bound in the starch granule. Following the report from Smith(reviewed above and in Martin and Smith, 1995) that pea contains a 76-KDagranule-bound starch synthase, Mu et al. (1994) made antibodies to a maizeendosperm granule-bound 76-KDa protein. These antibodies inhibited 90% ofthe maize endosperm SSS and precipitated a soluble protein of 76-KDa. Notonly are the results consistent with the conclusions drawn from the previouswork with the pea granule-bound starch synthase, but also these data point toa possible dual local ization of SS in the maize endosperm. It is not presentlyclear whether the non-Wx maize granule bound SS is in fact the same as themajor SSS , or whether the proteins share enough sequence similarity to causeantibody cross reaction. A preliminary report concerning the cloning of anon-Wx maize endosperm starch synthase has recently appeared (Ham et aI.,1995) . Hopefully, this will allow for a molecular determination of whetherthis granule bound starch synthase and the soluble SS are in fact identical.A mutant of the gene cloned by Wasserman et al. would certainly aid indistinguishing among the various possibilities.The data of Nelson et al. (1978) provide clear evidence for the presence of

the two starch bound SSs in the maize endosperm. Examination of a seriesof wx mutants uncovered a Wx-independent, low Km form of bound SS. Itseems likely that the 76-KDa protein isolated by Wasserman and colleaguesrepresents the Wx-independent starch syntha se reported previously from theNelson laboratory. We cannot rule out the possibility that both bound starchsyntheses are found in maize and peas, but their relative importance to amylosebiosynthesis differs in the two plants.Martin and Smith (1995) reviewed the various SSS activities in pea and

other plants and therefore this information will not be repeated here. As theseinvestigators emphasize, plant cells contain a series of starch syntheses, somebound and some unbound to starch granules. A certain portion of the lattermay also be found entrapped in starch granules as well.To summarize, the only mutant data so far available are derived from

wx mutants in maize and from a few other plants . These mutants provide


definitive evidence for the importance of the granule-bound starch synthasein amylose synthesis. In maize and other plants, the major bound activityresides with the smaller 59-KDa protein although there is some evidencethat a 76-KDa bound SS is also functional for amylose synthesis. Whileanalysis of wx mutants provides definitive evidence that amylose synthesisis via the 59-KDa protein, it is possible that the 76-KDa bound SS could beinvolved in amylopectin biosynthesis. Amylopectin amount and compositionare unaffected in mutants (reviewed in Nelson and Pan 1995); however, lossof granule bound activity affects amylopectin synthesis in Chlamydomonasreinhardtii (Maddelein et al., 1994). Perhaps the presence of two boundactivities in maize, but only one in C. reinhardtii, accounts for this difference.

Step 7. Starch branching enzymes and their genetic control

Multiple forms of starch branching enzymes (SBE) have been described inmany plant tissues. These enzymes are responsible for the alpha 1,6 bonds thatform the branches in amylopectin and phytoglycogen. SBEs differ in substratespecificity and the degree of branching produced (reviewed in Nelson and Pan,1995). As eluded to in the introduction, the very first mutant described for anyorganism (Mender's wrinkled pea mutant) affects starch synthesis, and studiesfrom Cathie Martin and associates (Bhattacharyya et al., 1990) showed thatthis mutant is defective in one of the pea SBEs.Three SBEs exist in the maize endosperm: I, IIa, and lIb (Boyer and Preiss ,

1981, Fisher et al., 1996). In other plants (Martin and Smith, 1995) , SBEsare usually placed in only two classes. Whether this reflects a fundamentaldifference in the various plants or tissues is not known; however, the geneticand molecular evidence in maize leads to the conclusion that more than twoSBEs are present in the endosperm. SBE I exhibits fundamental differences inpurification properties and enzyme kinetics compared to SBE lIs. While SBEIIa and lIb are nearly identical in purification properties and enzymologicalcharacteristics and share some sequence similarity (Singh and Preiss 1985),they are clearly different entities . Definitive insight came with the analysis ofthe amylose-extender (ae) mutations. As the name implies, mutants of thisgene increase the percentage of amylose in the mature maize endosperm.This occurs through a decrease in the amount of amylopectin synthesis. Also,the amylopectin that is synthesized is less branched than that found in wildtype. The se data provide no doubt that the Ae-controlled SBE is importantfor amylopectin biosynthesis.Does Ae encode SBE or does it control the amount of enzyme activi-

ty/protein by some indirect means? Two groups independently cloned Ae andshowed it is a structural gene for SBE lib. Fisher et al. (1993) cloned a genehomologous to a pea SBE, whereas Stinard et al. (1993) cloned the maizeAe gene through transposon tagging. Sequence comparisons showed that thesame gene was isolated. Clearly Ae encodes SBE lib.


Recently maize SBEI was cloned and the two cloned maize SBEs wereexpressed in an E. coli mutant lacking an endogenous branching activity(Guan et aI., 1995). The branched alpha-glucan synthesized in E. coli differsfrom that in wild type E coli, showing that the degree of branching is clearlya property of the SBE. Establishment of an E coli system to study starchsynthesis may help alleviate some of the confusion concerning the relativeimportance of the various SBEs in amylopectin biosynthesis. For example,while it was clearly established through mutant analysis that the Ae-encodedSBE IIb is important for amylopectin synthesis , in vitro enzyme assays typi-cally do not assign a predominant role to this enzyme (Guan and Preiss, 1993,and references cited therein) and enzymic properties inferred from in vitrostudies do not explain the types and amounts of polysaccharides found invarious maize single and double mutants (reviewed in Nelson and Pan 1995).

Step 8. Starch debranching activity and its genetic control

Debranching activity is important to normal starch synthesis. This surprisingconclusion comes from analysis of the sugaryl (sul) mutants of maize. Themutant form of this gene, long known and utilized in maize genetics andbreeding, conditions an increase in highly branched phytoglycogen (reviewedin Boyer and Hannah, 1994, Hannah et aI., 1993). Some mutant alleles (suchas sul-R, which is found in many conventional sweet corns) but not all,also increase sucrose amounts. A surprising outcome from an examination ofstarch synthetic enzymes was the observation that starch debranching activityis greatly reduced in sui mutants (Pan and Nelson 1985). In agreement withthis conclusion, James et al. (1995) cloned Sui through the use of Robertson 'sMutator and conventional transposon tagging. Since the cloning strategy wasindependent of any biochemical insight into the locus, finding significantsequence similarity of the Sui-tagged locus to a family of genes involved inthe cleavage of alpha 1,6 bond s provided independent confirmation of thework of Pan and Nelson (1985) for Sui being involved in the debranching ofstarch. These collective data show that starch debranching activity is involvedin starch synthesis. Perhaps it is not surprising then that E. coli enzymes thatsynthesize and degrade alpha 1,6 bonds are encoded by genes in the sameoperon (Preiss and Romero 1989) .Analysis of a double mutant involving su-R provided fundamental insight

into the role ofphytoglycogen in the starch biosynthetic pathway. Pan and Nel-son (1985) reviewed and emphasized the fact that phytoglycogen content isreduced in an ae sul-R double mutant compared to single sul-R mutants . Thisargues that the major branching enzyme (lIb) is important in phytoglycogensynthesis. Based on the logic expressed by Pan and Nelson (1985), phyto-glycogen and amylopectin are shown as interconvertible in Figure I. Accord-ing to this scheme, both branching and debranching activity are present inwild type developing endosperms. Functional debranching activity effective-


ly eliminates phytoglycogen synthesis in wild type endosperms. Only whendebranching activity is deficient (in sul mutants) does one observe a buildup in phytoglycogen. Since phytoglycogen is synthesized from amylopectin,loss of IIb branching activity greatly reduces phytoglycogen content.While the starch synthetic pathway put forward by Pan and Nelson (1985)

and summarized in Figure I accounts for the biochemistry of sui ae doublemutants, this scheme does not predict the amount of phytoglycogen in thesui btl double mutant (reviewed in Hannah et aI., 1993). Phytoglycogen isgreatly reduced in sui sh2 double mutants (as predicted by the pathway in Fig-ure 1), but the addition of a recessive bti allele to a sui-R background reducesphytoglycogen by only 50%, whereas starch content is greatly reduced. Thisis surprising since bti is thought to be involved in the transport of the C-6sugar/s (possibly ADP-glucose) and phytoglycogen is thought to be local-ized in amyloplasts. Two possible explanations come to mind . Perhaps thephytoglycogen synthesized in btl sul -R double mutants is actually in thecytoplasm. To my knowledge, there are no relevant data on this possibility.Alternatively, if Btl is also involved in formation of the primer for starch syn-thesis (reviewed in Nelson and Pan , 1995), then the reduction in the numberof functional primers might increase the relative amount of elongation (starchsynthase) and branching. This speculation makes the testable prediction thatstarch in bti mutants is longer than that found in wild type.There is one other obvious unknown concerning sui . How does loss of

debranching activity increase sucrose content? It is interesting that not allsul alleles increase sucrose. Some alleles increase phytoglycogen withoutincreasing sugar levels. Perhaps the SU I protein is multi-functional or perhapsthe Sui locus is more complex than we presently believe. There is certainlymuch more to be learned about this interesting gene/protein.

Tissue specific expression ofstarch synthetic genes

In studies of the maize mutants affecting starch biosynthesis (those listedin Table 1), visualization of the mutant phenotype is restricted to only a fewtissues , usually the endosperm. For example, mutation at Sh2 on chromosome3 conditions a severely shrunken or brittle kernel at maturity due to inadequateamounts of starch in the endosperm. No other tissue in sh2 plants exhibits analtered starch content. After identification of the biochemical lesion associatedwith sh2, AGP (Tsai and Nelson, 1966), subsequent studies measured AGPlevels in a number of tissues, including the embryo (Hannah and Nelson,1976), pollen (Bryce and Nelson, 1979) and leaves (Fuchs, 1977) . WhileAGP could be detected in all these tissues, only the endosperm enzyme wassubstantially reduced in sh2 mutants. These studies raised the interestingquestion of whether the same isoform of AGP is expressed in all these tissuesand Sh2 is a regulatory gene expressed only in the endosperm, or if differentisoforms of AGP exist in various tissues. Subsequent work showed that Sh2


is a structural gene for AGP (Hannah and Nelson, 1975, 1976; Bhave et aI.,1990) and that different forms of AGP exist in the various tissues (Preiss etaI., 1971; Giroux and Hannah, 1994, Prioul et aI., 1994).The Sh2:AGP relationship holds for all the other gene/enzymes listed in

Table I. A slight modification on this theme is found with the genes waxy (Wx)and amylose-extender (Ae). Mutations of these genes affect starch synthesis intwo tissues: the endosperm and the pollen. It is also interesting that these twogenes are distinguishable in another regard: they affect starch compositionrather than starch content. The other endosperm-specific genes affect starchcontent.

Tissue-specific mutant phenotypes do not prove tissue-specific geneexpression

Other variations on the Sh2 - AGP relationship exist. Mutation at the Shllocus gives rise to a noticeable phenotype only in the endosperm. However,molecular studies revealed that the Shl transcript can be detected in roots andshoots, particularly following growth under flooding conditions (Springer etaI., 1985, McCarty et al. 1986). While this does not prove the expression ofthe gene in these tissues (for example the transcript could have arisen from aclosely-related gene), these earlier studies employed a null or knock-out shlmutant to definitively identify the Shl transcript. The physiological relevanceof Shl expression in roots and shoots remains to be elucidated. It is possiblethat another enzyme system for this biochemical step operates in these tissuesand therefore genetic loss of Shl function does not condition a discerniblephenotype. These studies do make a point of major relevance to other studiesof biochemical genetics: identification of a mutant phenotype in only onetissue is not definitive evidence for tissue-specific expression of the gene inquestion.There are exceptions of the opposite type, namely cases in which loss of

gene activity in one tissue has a secondary consequence observed in othertissues. An example is found in the case of the Sh2 locus. While Sh2 onlyencodes AGP in the endosperm and different isoforms of this enzyme areexpressed in other tissues, consequences of Sh2 loss are seen in other tissuesand at different stages of plant development. Mutant sh2 seedlings and plantslack the vigor of their normal counterparts. This is a particularly importantproblem in the development of the new "super-sweet" sweetcorns. That tissuesother than the endosperm are affected by sh2 mutations bring into questionthe notion that the Sh2 gene is expressed only in the endosperm. However,recent results (Parera et aI., 1993 and Parera, Cantliffe, McCarty, and Hannah,submitted) have shown that seeds containing a wild type embryo, but a mutantendosperm, germinate as poorly as seeds containing both a mutant embryoand endosperm. These non-concordant seed were produced by use of BAtranslocation that contains a B centromere and a portion of chromosome 3 (the


A chromosome) containing the functional Sh2 gene. The chromosome non-disjoins at the mitosis giving rise to the two sperm and results in geneticallydissimilar male gametes . The results of this experiment are as expected if thepresence of a mutant endosperm somehow leads to damage of the intimately-associated wild type embryo .Were Sh2 expre ssed in the embryo and involvedin seedling vigor one would have expected restoration of vigor in the non-concordant seeds.

Leaky mutants , parallel biosynthetic steps , and epistasis

It is commonly observed in studies of starch synthetic mutants that the particu-lar defect under examination does not totally abolish all enzymic activity. Thisis true for all maize studies without exception. Two explanations exist: (1) themutant is leaky (the genetic lesion does not destroy all function of the gene)or (2) there exists another gene expressing the particular enzyme. While leakymutants clearly exist, whenever detailed examinations were made, a secondgene which encodes the particular enzyme was uncovered. An example ofthis is the genetic control of endo sperm sucrose synthase. A classic mutantgene of maize, shrunkenl (shl), conditions a greatly reduced level of thisenzyme (Chourey and Nelson , 1976). Examination of a series of mutant shlalleles revealed that none totally lacked sucrose synthase activity. Definitiveinsight came with the work from Burr and Burr (1981) who showed that oneof the "leaky" mutants was in fact a deletion. Clearly, the residual sucrosesynthase activity was under the control of a separate locus.The earliest example of this type of genetic redundancy can be found in

the case of the waxy (wx) locus. Thi s locus encodes a starch bound glucosyltransferase as previously described. In the seminal report of this finding(Nelson and Rines, 1962), enzyme activity in each of a series of wx mutantshad a Km for the substrate ADP-glucose ten-fold lower than that exhibitedby the wild type enzyme . These results clearly pointed to the existence of asecond starch granule bound starch synthase independent of the Wx locus.The analysis of extant mutants raises a number of interesting questions.

What would be the phenotype, for example, of a mutant totally lacking instarch synthesis? Would such a mutant be viable? If lethal, at what stage inendosperm development might arrest occur? It is interesting that the maizegenetic literature is filled with seed mutants classified as defectives. Whilethese mutants are sometimes viewed as developmental mutants, it is equal-ly possible that many represent lesions in the starch biosynthetic pathway.Clearly these mutants could represent an unmined treasure chest for futurestudies of starch synthesis.Biochemical redundancy for many if not all the steps in starch synthe-

sis exists in the maize endosperm. Moreover, the existence of more than onepathway is a distinct possibility. This complexity raises a number of importantissues . The interpretation of epistasis, for example, requires extreme caution.


Classically, epistatic interactions of non-allelic mutants have been used toorder the relative positions of steps within the pathway. If the phenotype ofthe double mutant resembles that of one of the mutant parents, conventionalwisdom states that the lesion associated with the phenotypically "dominant"mutant occurs before that of the "recessive"mutant. This type of logic can notbe used with leaky mutants or redundant pathways. For example, the enzymeencoded by the Shl gene acts before that of the Sh2 locus. However, the phe-notype of the shl sh2 double mutant closely resembles that of sh2 (Tracy,per.com.). This is readily explainable since the shllesion is phenotypically leaky,due to the presence of invertases and other enzymes that catabolize sucrose.The data definitively show that in cases of mutants lying in functionallyredundant biochemical steps, the concept of the "weakest link in the chain"is the more appropriate interpretation of the data. Such data cannot be used toorder biochemical steps. It is interesting to note that investigators interested indevelopmental pathways are currently using phenotypes of double mutants toorder unknown biochemical steps . Hopefully, some of complexities describedhere will be considered in the design and interpretation of their experiments.

Rate-limiting steps and dominant alleles

What step in a biochemical pathway is rate-limiting and is there only one?These are clearly important, if not the most critical, questions addressed byinvestigators seriously interested in elucidating biochemical pathways. Clas-sically, this question is addressed through biochemistry and enzymology.Identification of the first unique metabolite in the synthesis ofthe endproductand characterization of the enzyme synthesizing this molecule are typical firstapproaches. In the case of starch synthesis , it is now safe to conclude thatthe sugar nucleotide, adenosine diphosphate glucose (ADP-glucose), is themajor if not exclusive substrate for starch synthesis. The enzyme synthesiz-ing it, AGP, is allosterically affected, a property commonly associated withmetabolically-controlled enzymes. Definitive genetic evidence was presentedabove showing that AGP clearly does playa key, rate-limiting role in thesynthesis of starch/glycogen in organisms ranging from E. coli to the storageorgans of maize and potato.Are there other important, "rate-limiting" steps in starch synthesis? This

is completely unclear. One cannot safely conclude that there exists onlyone rate limiting step. For example, if the concentration of each metabolitein a pathway were equal to the Km of catabolizing enzyme, then geneticmanipulation of any enzyme in the pathway leading to the reduction of itsKm value would increase the flux through the pathway.Simple genetic data bear on the number of rate limiting steps. The vast

majority of physiologically significant mutations are detrimental , usuallyleading to a reduction or loss of gene function. Independent of this fact isthe observation that virtually all mutations are recessive. The phenotype of


the heterozygote is identical to that conditioned by homozygosity for thewild type gene. One copy of the wild type gene is commonly sufficient tocondition the wild type phenotype. Furthermore, the vast majority of genesshow a dosage effect at the enzyme level. In diploid tissues, the amount ofenzymic activity detected in vitro is usually approximately 50% of that seenin the homozygous normal individuals.A classic example of dominance and dosage effects can be found with the

waxy (wx) locus of maize, which encodes the starch granule-bound starchsynthase. Enzyme activity in the triploid endosperm containing 0, 1, 2, or 3doses of the functional gene showed a proportional increase with the numberof wild type genes (Tsai, 1965). Measurement of the endproduct, amylose,did not reveal this pattern. Amylose amounts in kernels with 2 and 3 dosesof the wild type gene were virtually identical. Amylose levels in kernels withone dose were nearly the same as kernels homozygous for the wild type gene.Thus, in the case of the Wx-encoded starch synthase, cells do not requirewild type enzyme levels to produce wild type amounts of amylose. The datasuggest that the amount of enzyme required is between 1/3 and 2/3 of thatfound in wild type. Evolution has allowed or selected for substantial bufferingcapacity in enzymic activity. Large reductions or increases in enzymic activitycan occur without significant effect on the amount of final product. Not only isthis fact important in elucidating rate-limiting steps, but also this observationis relevant to many physiological studies in which a particular treatment isfound to alter the amount of protein, enzyme or transcript. While observationscan be made and quantified , interpretation of data requires insight into thethreshold amount of the gene product needed to cause a physiological effect.Rarely is this known.Perhaps more interesting than the Wx-starch bound starch synthase rela-

tionship to the question of rate-limiting steps is the observation that levels ofAGP show a Sh2 and a Bt2 dosage effect yet one dose of the wild type geneconditions a kernel phenotype indistinguishable to the eye from three dosesof the functional allele. In other words, mutants of these loci are recessive ,yet AGP represents a rate-limiting step in starch biosynthesis. One mightexpect that kernels containing less than 100% wild type enzyme levels wouldhave a phenotype distinct from wild type. This is clearly not the case, at leastfrom visual inspection of the kernels. Conceivably, the in vivo responses toallosteric effectors might compensate for the lowered enzymic activity in theheterozygous genotypes. In other words, the in vitro enzyme measurementsdo not truly mimic the in vivo situation. However, G. Singletary and P.Keeling(per. com.) measured starch content throughout development in near-isogenicmaterials containing variable numbers of functional Bt2 alleles. Averagedover all developmental time periods, starch content per seed showed a linearincrease with the number of functional Bt2 alleles. This is expected if AGPregulates a rate-limiting step.


The foregoing discussion suggests that a search for dominant starch mutantsmight be a productive exercise. While dominant mutants are thought to beextremely rare events, since they are usually considered to be gain-of-functionmutations, loss-of-function mutations would appear as dominant or semi-dominant if a reduction in the wild type level of the enzyme in question givesrise to a visually detectable phenotype. Dominant mutants have, of course,the further advantage that they can be seen in a heterozygous condition.

Starch mutants in commerce

Application of the many extant starch mutants and the knowledge developedfrom them to agricultural and industrial uses are obvious and multi-faceted .The sweet com industry is totally based on starch mutants. While the classicalsweet com varieties employed the sui -R mutant allele of the Sui locus, thenewer sweet corns contain the sh2-R mutant or are doubly mutant for sui -Rand sugary-enhancer (see). The mutant se allele conditions a doubling ofsucrose content in a sui -R background (reviewed in Hannah et al., 1993).Theswitch to sh2-R and se arose from the advantage of higher sucrose contentin the consumed product (a developing ear). A disadvantage, however, ofthe newer sweet corns is a reduction in seedling vigor, commonly thoughtto be caused by low starch content in the mature kernel. Because high sugarlevels are desired early in kernel development (at the eating stage), but higherstarch content is useful late in development, the addition of a Sh2 codingregion attached to a late-expressed endosperm promoter may provide a usefulsolution for sweet com breeders.Conversely, variants with higher starch content or different types of sugar

polymers have obvious applications. Since the mature maize seed is approxi-mately 70% starch, increases in starch content may also increase seed weightand hence yield . The use of altered AGPs in potato and in maize was reviewedabove .Altered types of starches conditioned by wx and ae mutants have been in

the market place for many years .Wx mutant starch is completely amylopectin,while that in ae has a much higher percentage of amylose. These starches areused in the synthesis of different types of industrial products . One obviousline of investigation is the development of maize endosperm totally devoidof starch branching activity. Data point to at least three independent genesencoding BE. Triple loss-of-function mutants should synthesize only amy-lose. This has obvious application, since millers would prefer to mix amyloseand amylopectin in the ratios they desire . Presently, the polymers must beseparated before they can be used for manufacture and processing.Generation of such triple mutants is now feasible. Investigators at Pioneer

Hi-Bred have generated large populations containing Robertson 's Mutator .Preliminary analysis suggests that every gene within the maize genome hasbeen tagged in this population. Using automated DNA extraction procedures


and peR, it is now feasible to identify Mutator insertion mutations for everycloned gene. This will greatly facilitate progress in isolating mutants which(1) are lethal in homozygous condition (the Pioneer procedure will identifymutants in heterozygous condition) and (2) have no discernible phenotype.The latter is predicted for single mutants lacking only SBEI or SBEIIa forexample.Reports describing the production of transgenic plants with alterations

in the types of polysaccharides produced are beginning to be seen in theliterature . Descriptions of potatoes producing fructans have appeared, andanalogous work in maize will appear shortly. This area of research providesunlimited opportunities, especially for production of industrial starches. Itis envisaged that soon "com" will not be an adequate term to describe thevarious types of maize that will appear in the market place.

Future areas of research

This chapter has emphasized the genetic approach to the study of starchsynthesis.Much progress has been made, and the primary biochemical lesionsassociated with many of the maize endosperm search mutants have beenelucidated. With the possible exception of Mnl, the genes have turned out toencode enzymes in the biosynthetic pathway.While there is now an impressive array of mutants, it is fascinating that

none of the genes turned out to encode a DNA binding protein or a proteinkinase. Deciphers of other pathways have frequently found such regulatorygenes, and it is intriguing that none have been found for starch synthesis.The significance of this is not obvious. Emerging evidence shows that at leastone of the starch biosynthetic enzymes, sucrose synthase (Shaw et al., 1994),is phosphorylated and the phosphorylation state of the protein affects theKm for sucrose (S. and J. Huber, per. com.). If these changes in the kineticproperties of sucrose synthase are physiologically relevant, one might expectto find a knock-out mutant for the sucrose synthase kinase. Perhaps suchstarch biosynthetic regulatory genes are functionally redundant, or perhapshomozygosity for null mutants is lethal. Leaky mutants have been found forvirtually every structural gene in the pathway, and therefore one would expectleaky regulatory mutants as well. This line of thinking would suggest that theformer hypothesis is more likely.This review has emphasized the fact that every important starch biosynthet-

ic enzyme expressed in the maize endosperm exists in at least two isoforms.Removal of the major sucrose synthase (mutation at Shl), major ADP-glucosepyrophosphorylase (mutation at Sh2 or 8t2), major branching enzyme (muta-tion atAe), or major starch bound ADP-glucose glucosyl transferase (mutationat Wx) uncovers a minor isoform of the enzyme. It is interesting to considerwhat the phenotypes of the mutants listed above might be if the minor isoformdid not exist. Indeed, it is conceivable that some of these mutants would be


homozygous lethals in the absence of the minor isoenzyme. It seems possi-ble that some existing homozygous lethal mutants may prove interesting indeciphering the regulation of starch synthesis .There is much left to do with the extant mutants. Why do some but not all

of the sul mutant alleles accumulate sucrose? Is Mnl really a structural genefor the cell wall invertase and, if so, why are the other invertases affected?Why do sul btl and sul bt2 mutants exhibit such fundamental differences inphytoglycogen content? Aren't Btl and Bt2 in adjacent steps in a sequentialpathway? Is AGP really located both in the cytoplasm and the amyloplasts?Are there other rate limiting steps besides AGP?How are the various pathwaysexpressed at high levels in the maize endosperm coordinated? Starch mutantsaffect the synthesis of the storage proteins or zeins. While zein proteins arereduced in the severe starch mutants, transcript levels are increased (Girouxet aI., 1994).Does all carbon in the path from sucrose to starch pass through the same

pathway? Nelson and Pan (1995) point out that this may not necessarily bethe case and hypothesize that the starch found in sh2 and bt2 mutants may besynthesized via phosphorylase. There is also reason to believe that not all ofthe sucrose entering the seed must be broken down and then resynthesized asthe proposed pathway (Figure 1)would suggest (Hannah et al., 1993).Nelsonand Pan (1995) raised a number of other important questions concerning thispathway that are yet to be resolved.

In the future, I suspect that the collection of Mutator -induced mutants atPioneer will prove invaluable in answering many outstanding questions. Thereverse genetics approach will allow the isolation of phenotype-less, loss-of-function, and homozygous lethal mutants for any cloned gene. This, coupledwith commercial interests in the pathway and the realization of people in acad-eme that there remain many interesting fundamentally-important questionsto resolve should provide further insight into this important and interestingpathway.

Acknowledgment

I thank Oliver Nelson, Brian Larkins, Martha James, Peter Keeling, JackShannon, George Singletary, and Tom Sullivan for sharing unpublished dataand for valuable critiques of an earlier version of this review. I thank DonMcCarty, Karen Koch, and especially Oliver Nelson for many insightfuldiscussions concerning starch synthesis in maize. Research in this laboratoryis supported by NSF grants, IBN-93 16887 and MCB-9420422 and USDACompetitive Grant , 94-37300-453. This is Florida Agricultural ExperimentStation Journal Series Number R-04930 .


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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Starch Synthesis in the Maize Seed