In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

Several thousand plant species, including many economically important food plants, synthesize cyanogenic glycosides and cyanolipids. Upon tissue disruption, these natural products are hydrolyzed liberating the respiratory poison hydrogen cyanide. This phenomenon of cyanogenesis accounts for numerous cases of acute and chronic cyanide poisoning of animals including man. This article reviews information gathered during the past decade about the enzymology and molecular biology of cyanogenesis in higher plants. How compartmentation normally prevents the large-scale, suicidal release of HCN within the intact plant is discussed. A renewed interest in the physiology of these cyanogenic compounds has revealed that, in addition to providing protection for some species against herbivory, they may also serve as storage forms for reduced nitrogen.Cyanogenesis, the ability of plants and other living organisms to release hydrogen cyanide, has been known for several centuries. Since its first description in plants in 1803 (17). It is highly desirable that the toxicity of cyanogenic plants to humans and livestock be reduced. This is achievable by: (a) selective breeding to produce low-cyanogen varieties, as was accomplished for almonds, (b) screening of natural populations for low-cyanogen varieties, (c) mutagenesis of protoplasts or cell cultures with subsequent regeneration of plants having desired mutant genotypes, or (d) genetic engineering. To facilitate these approaches, one must first acquire a working knowledge of the biochemistry, physiology, and molecular biology of cyanogenesis. This article provides an overview of recent publications which have advanced our understanding ofthese critical areas. Attention will be focused primarily on cyanogenic glycosides, but significant developments in the less studied field of cyanolipids are noted. BIOSYNTHESIS OF CYANOGENIC GLYCOSIDESMost cyanogenic glycosides are derived from the five hydrophobic protein amino acids tyrosine, phenylalanine, valine, leucine, and isoleucine. Cyclopentenoid cyanogens presumably originate from cyclopentenylglycine. A biosynthetic pathway originally proposed on the basis of in vivo tracer experiments has been confirmed by extensive studies with cell-free systems (4). Microsomal preparations from sorghum, arrow grass, linen flax, and Trifolium repens convert specific amino acids to their corresponding a-hydroxynitriles with Nhydroxyamino acids, aldoximes, and nitriles serving as intermediates (Fig. 1). The highly channeled nature of these pathway intermediates explains the failure of early labeling studies to detect them. The final step in glycoside biosynthesis is glycosylation of a-hydroxynitriles by soluble O-glycosyltransferases. These UDP-sugar dependent enzymes have been partially purified from sorghum, arrow grass, and black cherry.The most elaborate studies on cyanogenic glycoside biosyn-

The tissue distributions of dhurrin lphydroxy-(S)-mandelonitrhie8-Dplucosidel and ofenzymes involved in its metaboUsm have been investigated in leaf blades of light-grown Sorghu bicolor seedlngs. Enzymk digestion of these leaves using celulase has enabled preparations of epidermal and mesophyli protoplasts and bundle sheath strands to be isolated with only minor cross-contamination. Dhurrin was located entirely in the epidermal layers of the leaf blade, whereas the two enzymes responsible for its catabolism, namely dhurrin ,-glucosidase and hydroxynitrile lyase, resided almost exclusively in the mesophyli tissue. The final enzyme of dhurrin biosynthesis, uridine diphosphate glucose;p-hydroxymandelonitrile glucosyltransferase, was found in both mesophylH (32% of the total activity of the leaf blade) and epidermal (68%) tissues. The bundle sheath strands did not contain significant amounts of dhurrin or of these enzymes. It was concluded that the separation of dhurrin and its catabolic enzymes in different tissues prevents its large scale hydrolysis under normal physiological conditions. The well documented production of HCN (cyanogenesis), which occurs rapidly on crushing Sorghum leaves, would be expected to proceed when the contents of the ruptured epidermal and mesophyll cells are allowed to mix.

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