Discoveries in Plant Biology

The following sections are included:

• PREFACE

• CONTRIBUTORS

• CONTENTS

An account of the development of knowledge of the elements essential for the growth and development of higher plants is given. It was only since the end of the 18th century that the very concept of chemical elements and chemical reactions was understood. It took a further half-century before plant scientists became convinced that chemical elements go into the makeup of plants, and that most of these are mineral nutrients absorbed by roots. By now, in addition to carbon, hydrogen and oxygen, the number of elements known to be essential for higher green plants is 14; three more are essential for at least some plants, or under certain conditions. The discovery of the essentiality of elements depended upon refinements of the solution culture technique, the only method of withholding an element required by plants in very small amounts to an extent where its deficiency becomes apparent.

This chapter provides an account of how 1-aminocyclopropane-1-carboxylic acid (ACC) came to be recognized as the immediate precursor of the plant hormone ethylene. This discovery relied heavily on earlier observations and technological advances in the field, such as the introduction of gas chromatography with flame ionization detection as a rapid and sensitive analytical method for the gas. Likewise, the discovery that methionine is a close precursor was an important pre-requisite for complete elucidation of ethylene's biosynthetic pathway. The first step in establishing the pathway from methionine to ethylene was obtaining evidence that S-adenosylmethionine was an intermediate. This was done by showing that 5'-methylthioadenosine and methylthioribose were products of methionine metabolism in ethylene-producing tissues. A brief explanation is given of the role in the discovery played by a class of inhibitors, 3,4-unsaturated amino acids, the most important member of the class being aminoethoxyvinylglycine (AVG). The way that these inhibitors were used to establish the order of intermediates in the pathway is summarized. This account describes how feeding studies using radioactive methionine under anaerobic conditions led to direct observation of a labeled metabolite which fit the criteria for a hypothetical intermediate that had been proposed by several previous investigators. The experiments undertaken to identify the unknown metabolite (ACC) are described; the result being that this unusual amino acid, which had been isolated from apples and pears twenty years prior, was finally recognized as the immediate precursor of ethylene.

Pioneer studies in the 19th – early 20th century leading to the discovery of auxin are introduced first. In 1928, Dutch botanist Fritz W. Went finally isolated auxin diffused out from the tip of oat coleoptiles in the gelatin block. Following Went's success, auxin, indole-3-acetic acid (IAA) was then isolated first from human urine, then from fungi, and finally from higher plants. Discovery of auxin is thus the result of the work of many botanists and organic chemists from various countries. Biosynthesis, metabolism and physiological actions of auxin are also briefly described.

The non-reducing sugars floridosides and sucrose, which are abundant in red algae and green plants, respectively, are the initial stable neutral photosynthetic products. Although all three isomers of floridosides, or α-D-galactosylglycerols, are known, their metabolic roles are still under speculation. By radiotracer feeding studies, we demonstrated a cycling of sucrose synthesis cleavage without net gain of sucrose, and the glucose moiety of sucrose was preferentially incorporated into the rice seed starch. Sucrose synthase (SuS) has been implicated as the key enzyme in both systems. The enzyme is common in green plants, and consists of four identical or mixed protomers. Three rice SuS genes were cloned and their structures mapped. One of the isologous genes is expressed in the endosperm of starch-filling rice seed, and the other two are ubiquitously expressed, with one dominating over the other in different tissues. Other sucrose-metabolizing enzymes, invertase and sucrose phosphate synthase, should play distinctly different physiological roles because they catalyze opposite irreversible reactions. Man regard sucrose as the most important natural sweetener of all time. Not many enzymes are directly involved in the metabolic pathways spanning around it. However, further investigations on the exact mechanisms by which sucrose is used as carbon and energy sources and as a metabolic regulatory signal in the maintenance of plant life are still required.

Biochemical, genetic and molecular adventures have resulted in never-ending, exciting discoveries in the elucidation of the chlorophyll biosynthetic pathway from 1945 to 1997. Major developments are traced in this chapter. Special emphasis is placed on the pathway to 5-aminolevulinate by David Shemin, on the information gained from Sam Granick's mutants in Chlorella and from the protochlorophyllide holochrome by James H. C. Smith. Analysis of structural and regulatory mutants in higher plants and photosynthetic bacteria support the generality of the pathway. The bacteriochlorophyll operons of Barry Marrs and John Hearst together with the evolving techniques of nucleotide and protein sequencing, as well as those for crystal structure analyses and site-directed mutations have opened the way to discoveries, how the different enzymes accomplish their task in synthesizing chlorophyll — an indispensable molecule for life on earth. Antisense gene technology teaches us how the plant avoids destruction by light-sensitive intermediates. It has been a privilege to have been able to follow the adventures of many scientists mentioned in this and the following review and to interact with a significant number of them. I am also grateful to the many enthusiastic and skilled students, postdoctoral fellows and colleagues, who have allowed me to participate in their discoveries.

Biochemical, genetic and molecular adventures result in never-ending, exciting discoveries in the elucidation of the chlorophyll biosynthetic pathway. This is exemplified by the discovery that Shemin's single enzyme pathway for the 5-aminolevulinate precursor of chlorophyll is only used by some photosynthetic bacteria. Cyanobacteria, algae and all plants use a multi-enzyme pathway involving a unique step of activation with a tRNA. Most fascinating is the variety of enzymes and mechanisms photosynthetic organisms employ for the same step in the pathway. This chapter highlights interesting biochemical and molecular aspects of fourteen steps in the biosynthesis of chlorophyll.

Higher plants possess two independent pathways for isopentenyl diphosphate (IPP) and isoprenoid formation that work in different cellular compartments and deliver differential isoprenoid end products: 1) in the cytosol proceeds the classical acetate/mevalonate pathway for the biosynthesis of sterols and sesquiterpenes, and 2) in the plastids the non-mevalonate l-deoxy-D-xylulose-5-phosphate pathway (DOXP-pathway¹) for the biosynthesis of carotenoids, phytol, prenyl side-chains of plastoquinone-9 and phylloquinone K₁, as well as isoprene and mono- and diterpenes. The acetate/mevalonate pathway of plants had already been established in 1958. However, the DOXP-pathway was only elucidated in the past four years, although various labeling results of plastidic isoprenoids were not in agreement with the mevalonate pathway. These controversial issues and the steps that finally led to the discovery of the plastidic DOXP-pathway of IPP and isoprenoid formation are summarized in this chapter.

Studies on both the structure and mechanism of synthesis of cellulose, the world's most abundant organic compound, have been challenging and often controversial. Cellulose got its name by being the “sugar” of plant cell walls. It was first isolated and characterized as an aggregation of glucose units by Anselme Payen 160 years ago. Knowledge of its structure has developed over time, along with many of the concepts of carbohydrate and polymer chemistry. Some major landmarks include an appreciation for the complexity of the structures of cellulose in cell walls by Balls, and that cellulosic fibers are composed of particles or micelles that we now call microfibrils. On a molecular basis, the determination by Polanyi of the unit cell for the most prevalent native form and the subsequent refinements of the unit cell and the molecular structure by Meyer and colleagues were major findings. Their reports were interspersed with the realization that the glucose monomers were rings of six atoms instead of five. That development enabled correct interpretation of the methylation data regarding the β-1,4 linkages between glucose residues. It also allowed a revision of Tollens' earlier idea that cellulose was a long chain of covalently joined glucose units rather than an aggregation held together by secondary forces. Cellulose is polymorphic. The chains apparently pack in both parallel and antiparallel arrays, and different backbone shapes are found for different derivatives and complexes. Several different forms are found for native plant systems. Elucidation of the exact details of these different forms is a current research area. To date, the best results are available for crystalline cellodextrins which have permitted full-scale, single-crystal X-ray diffraction determinations.

In reviewing the history of research on cellulose biosynthesis, one must first acknowledge the pioneering work on the bacterium — Acetobacter xylinum — first initiated by Shlomo Hestrin in Israel. Through the years, A. xylinum has been developed as a model system for the first demonstration of in vitro synthesis of cellulose using UDP-glc as substrate, to identify a unique activator of the cellulose synthase cyclic diguanylic acid, to purify the first synthase, and to clone an operon of genes that encodes essential components of the synthase complex. Studies with algae and higher plants using freeze-fracture techniques have revealed multisubunit cellulose synthase complexes in the plasma membrane. However, at the molecular level, studies with plants have always lagged behind those of A. xylinum. Nevertheless, the work of many groups has gradually provided insight concerning the difficulties of in vitro synthesis and enzyme purification in plants as well as an understanding of motifs that are conserved in glycoyltransferases that use UDP-glc as substrate. Understanding of these conserved motifs was a key to the recent success in the first cloning of a gene that encodes the plant catalytic subunit of the synthase complex. Taken together, much clearer views of both the structure and mechanism of synthesis of this polymer are now emerging.

Although the basic studies in starch biosynthesis were carried out in England during the 1940s and led to the discovery of phosphorylase and Q enzyme (branching enzyme), the basis of our modern ideas originated in Argentina from the work of Luis F. Leloir and Carlos E. Cardini. During the late 1950s, they established that nucleoside diphosphate glucose was involved in the synthesis of both glycogen and starch. In this paper, we describe the events leading to these discoveries and the implications of that early research for starch biochemistry as well as for plant biochemistry in general and our understanding of human and bacterial metabolism.

Plant seeds are a rich food source. They contain high levels of carbohydrate, lipid and protein; nutrients that are essential for human growth and development. A major class of proteins found in seeds are the storage proteins. These proteins (usually) have no enzymatic or structural function other than to simply serve as a nitrogen, sulfur and carbon reserve for the post-germination phase of growth in the young developing seedling. As a result of their nutritional value and their importance in breadmaking, the storage proteins have been studied ever since the 1700s. In this chapter, we present a historical overview on the nature of the storage proteins from different plants with special reference to their structures, evolution, and cellular processes that are responsible for their synthesis and packaging in the cell.

Seeds contain abundant storage proteins as reserve organic nitrogen for germination and postgerminative growth. In the early 1970s, it was thought that in seeds other than the cereals, water insoluble globulins of high molecular weights were the storage proteins, whereas water soluble albumins were metabolic proteins such as enzymes. A series of reports then established that low molecular weight albumins of 2S sedimentation coefficient represent a major group of seed storage proteins, comparable in quantity and ubiquity to the globulins. The 2S albumins are localized in the protein bodies and rapidly degraded during germination and postgerminative growth. They possess high contents of nitrogen-rich amino acid residues and the sulfur-containing methionine and cysteine as reserves of organic nitrogen and sulfur, respectively. They are the seed allergens that had been studied previously. Their high content of the essential amino acids methionine and cysteine make them desirable nutrients for human and animal feed. Subsequent studies by different laboratories have defined the structure of the 2S albumins, the proteolysis of the nascent polypeptide into the final products, the targeting signals in the flexible molecule to the protein bodies, the characteristics of their genes, the inclusion of the cereal prolamins and enzyme inhibitors in the seed 2S albumin family, and the evolution of the whole gene family. Genetic engineering of the 2S albumins has included incorporation of their genes into seed crops to enhance the essential amino acid contents, manufacture of pharmaceutical polypeptides using the abundant 2S albumin as a carrier, and employment of their active seed-specific gene promoters for different purposes.

My involvement in the study of Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), goes back almost thirty years ago. In 1971, I joined Dr S G Wildman's Laboratory in UCLA. Wildman's laboratory was, at that time, heavily engaged in the study of various chemical components of chloroplasts, such as chloroplast DNA (ct-DNA) and chloroplast proteins. Therefore, my PhD training on ct-DNA in Toronto qualified me to join his group. I studied the physico-chemical properties of ct-DNA in the University of Toronto and published a couple of papers in BBA (Kung and Williams, 1968 and 1969) which caught his attention. He offered me a position as a research associate, a super post-doctoral fellow, since I had already finished two years' post-doctoral training in the Biochemistry Department, The Hospital for Sick Children, Toronto. Although my PhD degree was from the department of Botany, my work was entirely in the area of biochemistry and biophysics. The training in plant physiology, biochemistry and biophysics proved to be a commendable advantage in advancing my career.

Cytochromes were first shown to be present in chloroplast membranes of higher plants in the laboratory of Robin Hill in Cambridge. Cytochromes f and b-563 were subsequently shown to be present in a cytochome bf complex, which catalyzed electron transfer between plastoquinol and plastocyanin, whereas cytochrome b-559 was located in photosystem II, where its function is still unresolved. The purification of the proteins led to the identification of the heme-binding polypeptides, and it was established that all of the chloroplast cytochromes were synthesized within the chloroplasts. The genes encoding the heme-binding polypeptides of each of the cytochromes were located in chloroplast DNA, and structural models of heme binding and transmembrane arrangement were proposed based on the protein sequences of the cytochromes. These models of the overall structures of the cytochromes will soon be tested rigorously, in the light of recent and future crystal structures of cytochrome components.

Studies on the direct role of ATP in providing energy for the work of mineral nutrient absorption in plants led to the discovery of plasma membrane-associated adenosine triphosphatase (ATPase). The research was initiated by Tom Hodges during the late 1960s at the University of Illinois and continued during the 1970s at Purdue University. By 1972, methods for isolating plasma membrane vesicles from broken plant cells had been developed and used to characterize the associated ATPase activity. Over the next 15 years, the plant plasma membrane ATPase was shown to have many properties in common with analogous ion pumps in animal and fungal cells, including conserved DNA sequences successfully utilized for cloning plant ATPase genes. The initial hypothesis that plasma membrane ATPase directly participated in K⁺ absorption by root cells was not supported by research results. Rather, this ATPase is the electrogenic proton pump that establishes and maintains a proton-free energy gradient across the plasma membrane to which energy-dependent ion and metabolite transport is coupled.

In just the last decade, proteolysis has finally assumed its rightful place as a key regulator of cell metabolism, growth, and development. One of the central players in this process in eukaryotes is ubiquitin, a highly conserved, 76-amino-acid protein. Ubiquitin functions as a reusable signal for selective protein degradation. Via an ATP-dependent cascade of reactions, multiple ubiquitins are attached to proteins destined for breakdown; the modified proteins are then selectively degraded to amino acids by the multi-subunit 26S proteasome with release of the ubiquitin moieties in free, functional forms. Here, I provide a historical perspective of our understanding of intracellular proteolysis and ubiquitin with an emphasis on their roles in higher plants. As will be seen, determining their functional importance required the concerted effort of a number of scientists working in many model systems. Insomuch as we have only scratched the surface of this fascinating aspect of biological control, the review should be read more as a prelude than a final appraisal.

The author summarizes the research contributions to photosynthesis made by him, his graduate and postdoctoral students, visiting scientists and by his collaboration with other photosynthesis workers during 1964–1994. The development of isolation procedures and biochemical/biophysical characterization of antenna pigment-proteins and photochemical reaction centers are described together with the author's education and experiences as a scientific researcher. Some anecdotes hopefully add insight into what it was like to be in this area of science during the period.

When soybean seedlings or cultured cells of soybean and tobacco are shifted from a normal growth temperature of 28°C to higher temperatures, the synthesis of a new set of proteins (heat shock proteins, or HSPs) is induced while the synthesis of most proteins made at 28°C is reduced, as first reported by J. L. Key, J. P. Mascarenhas and their co-workers in 1980. A number of other plants have since been shown to respond similarly to heat shock as soybeans do. This response in plant systems seems to have many properties in common with the heat shock response of Drosophila and other eukaryotic systems. The pattern of heat shock protein synthesis in soybean seedlings is closely paralleled by the accumulation and subsequent loss of heat shock-specific mRNAs. The acquisition of thermotolerance in plants correlates well with the presence of HSPs. Following the first reports of plant HSPs in the early 1980s, multifamilies of heat shock genes have been isolated and studied in different plant systems. The localization of heat shock proteins during stress and their roles in thermotolerance have also been studied. The heat shock elements of plant genes and their expression in transgenic plants have been described. Some HSPs in plants are also present during certain developmental stages, and some HSPs are induced by other stress agents. Recent studies of the heat shock response in plants have led to a better understanding of the heat shock system of plants at the molecular level, and of the possible physiological and biochemical significance of heat shock proteins in plants.

Light exerts a profound influence on the growth and development of green plants. Many of the developmental responses of plants to light are initiated by activation of the photoreceptor phytochrome. In this chapter, emphasis is given to the pioneering studies establishing that light acting through phytochrome triggers differential expression of specific nuclear genes.

Studies of the nitrogen fixation (nif) genes began in the Laboratory of Molecular Genetics in 1974. I was privileged to work with a group of young scientists from different areas of biology and chemistry, on what was regarded a fancy subject in those days. Results of fine-structure mapping and complementation tests of nif genes demonstrated that the nif genes of Klebsiella pneumoniae reside as a single cluster near the histidine operon (his) on the chromosome. Intragenic complementation occurred between two niff mutants, indicating the niff product as a dimeric protein of identical subunits.

For study of the regulation of nif genes, a test system for the role of nifA product NifA on nif gene expression was created. NifA was proven to be the key protein in the regulation of nif genes. There was evidence that the repressive action of the nifL gene product, NifL, is in its interaction with NifA. A two-level regulation hypothesis for NifA production has been proposed. Under oxygen, the transcription of nifLA operon is repressed, and NifA produced will be inactivated by its interaction with NifL. This hypothesis is now substantiated by experiments.

In Rhizobium meliloti, nod/nif genes were shown to express sequentially during the development of the rhizobia. NifA functions both for nodulation and nitrogen fixation.

In 1907, Smith and Townsend identified Agrobacterium as the causative agent of crown gall, the most common form of neoplasia in plants. Armin Braun, elaborating on Smith's idea about infectious plant cancer, predicted during the early fifties that Agrobacterium transfers a Tumor Inducing Principle (TIP) into plants that incites the proliferation of crown gall tumors by triggering the autonomous synthesis of plant growth hormones auxin and cytokinin. Also in 1970, Morel's group in France suggested that TIP is an Agrobacterium-derived DNA that specifies the production of unique compounds in crown galls termed opines that are characteristic for and catabolized by the tumor-inciting bacteria. That the TIP is indeed carried by a large tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens was demonstrated in 1973 by my research group in Gent, Belgium. We and others later showed that TIP corresponds to a segment of the Ti plasmid which is transferred by agrobacteria and stably integrated into the nuclear genome of plant cells. The transferred DNA (T-DNA) was found to encode eukaryotic genes required for the production of opines and plant hormones in crown galls. Inactivation of the T-DNA encoded oncogenes involved in hormone synthesis allowed us in 1981 to obtain fertile transgenic plants showing stable Mendelian inheritance of a T-DNA encoded opine synthase gene. These results opened the way to broad range exploitation of T-DNA in plant transformation, physiology and genetics.

The major principles of active transport were first elucidated in plants. These include the principles (a) that ions may enter an organism against their concentration gradients and independently of water movement, (b) that the processes of diffusion and osmosis are inadequate to sustain the observed ion gradients, (c) that ion movement depend upon the generation of metabolic energy, (d) that ion movement is carrier-mediated, (e) that electrochemical gradients across biological membranes drive ion transport, and (f) that at least two types of mechanisms are involved in the transport of virtually all ions. Plant materials such as giant algal cells remain model systems for ion transport studies.

It must have been obvious to farmers for millennia that plants differ in their sensitivity to low temperatures. Freezing damages most growing plants, but some plants are also injured by exposure to low, non-freezing temperatures. These chilling-sensitive plants are among the most important agricultural crops grown today and are major contributors to world food and fiber production. A partial list includes bananas, beans, citrus, cotton, maize, melons, millet, peppers, rice, sorghum, soybeans, squash, sugar cane, and tomatoes. The development of cultural practices from antiquity to the pre-scientific age that alleviated the severity of chilling injury symptoms indicates that the temperature limitations of many chilling-sensitive crops were well known. These practical methods to mitigate chilling injury far predate the scientific studies that were undertaken only during the past century to identify the physiological cause of chilling. The dominant paradigm for the past 25 years involves a causative progression from temperature-induced membrane phase transitions, to altered metabolism, to visual chilling injury symptoms. Much has been discovered about the practical effects of chilling, but the underlying molecular and physiological causes remain elusive and still awaits discovery.

Haploids have attracted the interest of plant physiologists, embryologists, geneticists and breeders since the first discovery of haploid plants in Datura stramonium as early as (Blakeslee et at., 1922). In the beginning, haploid plants were regarded as a special biological phenomenon. Up to 1960s, the occurrence of haploids were reported in 71 species of angiosperms belonging to 39 genera and 14 families (Kimber & Riley, 1963). However, the low frequency of spontaneously arising haploid plants severely limited the utilization of haploids for crop improvement and genetic studies. In the last three decades, many efficient and simple techniques, especially in vitro culture have been developed to produce haploid plants in larger numbers. For instance, by using anther culture, haploids were induced in 247 species of angiosperms including some hybrids, which belong to 88 genera of 34 families (Maheshwari et at., 1983). Meanwhile, chromosome elimination (the bulbosum technique), and in vitro culture of unfertilized ovaries and ovules have been proved to be an efficient means of haploid induction in some species. These advancements opened up the way for studying and utilizing the haploids in higher plants.

This paper mainly discusses these advancements of haploids in higher plants in terms of its origin, genetics and application in breeding.

The following sections are included:

• INDEX