Cloning and Identification of Highly Expressed Genes in Barley Lemma and Palea

expressed in the lemma/palea would provide molecular clues to the functions of these organs. The lemma and palea (lemma/palea) of cereals are photosynthetic Loss-of-function studies and cloning of floral homeoorgans that supply the developing kernel with carbon and nitrogen. tic genes suggest that the lemma/palea of grasses are Because of their rigid structure, the lemma/palea can also protect the developmentally analogous to sepals (Bowman, 1997; kernel from pathogens and herbivory. However, very little is known about specific gene expression that enabled the lemma/palea carry Ambrose et al., 2000; Prasad et al., 2001). In maize (Zea out their functions. We have constructed three subtracted cDNA mays L.), the male sterile mutant silky1 (si1), encodes libraries from lemma/palea of barley (Hordeum vulgare L. cv. Morex) a MADS box gene orthologous to the Arabidopsis AP3 at the elongation (between pollination and milky stages) through (a B class organ identity gene; Ambrose et al., 2000). dough stages of kernel development. Differential screening and northMutations in the si1 locus result in the conversion of ern hybridization showed that the cloned genes were highly expressed lodicules into lemma/palea. Since lodicules are the in the lemma/palea, compared with the flag leaf. Thus, they contained equivalent of petals (specified by class A and B genes), unique sequences not found in the flag leaf or were expressed in the replacement of lodicules by lemma/palea in si1 mutants lemma/palea at much higher levels, appearing as if they were induced. indicates that lemma/palea are specified by the class A Sequence analysis of 226 clones identified a high proportion of genes genes, as are the sepals (Ambrose et al., 2000). Direct for defense, structure, amino acid biosynthesis, and photosynthesis. evidence for the involvement of class A genes in the High expression levels of defense-related genes strongly suggest that lemma/palea constitutively accumulate defensive molecules to inhibit specification of lemma/palea is the cloning of cereal invasion of florets and kernels by pathogens. Increased expression of genes orthologous to the Arabidopsis AP1. The rice genes involved in cell wall synthesis and structural repair can improve (Oryza sativa L.) OsMADS1 gene is expressed early in physical barriers to herbivores and pathogens. High expression of the spikelet meristem, and later its expression is congenes for amino acid biosynthesis and photosynthesis indicates that fined to the lemma/palea primordia (Prasad et al., 2001). the lemma/palea are major sources of nitrogen and carbon for the This expression pattern is consistent with the early exgrowing kernel. pression of AP1 in Arabidopsis floral meristem and later in sepal primordia (Bowman et al., 1993; Parcy et al., 1998). RAP1A, also a rice ortholog of AP1 gene, is C inflorescences are organized into spikelets, expressed early in flower development; later its expresand each spikelet contains one or more florets sion is confined to the developing lemma/palea and lodi(Briggs, 1978). In turn, each floret consists of a pair of cules (Kyozuka et al., 2000). glumes, a lemma, a palea, a pair of lodicules, three Although sufficient evidence exists to show that floral stamens, and a pistil. The lemma bears a flower at its homeotic genes specify lemma/palea, no study has been axil and, later, with the palea forms the husk (Fig. 1). undertaken to determine which genes are active in maThe lemma/palea play indispensable roles in the develture lemma/palea. Expression analysis would enable us opment of cereal seeds. These photosynthetic organs, to identify genes that contribute to the functions of together with the flag leaf, and upper stem, supply flolemma/palea. Here we used the PCR-based suppression rets and developing kernels with carbohydrates (resubtractive hybridization (SSH) method to identify genes viewed in Duffus and Cochrane, 1993). The lemma/ overexpressed in mature lemma/palea (from elongation, palea and other photosynthetic organs of the spike conbetween pollination and milky stages, to early dough tribute up to 76% of kernel dry weight. Amino acids stages of kernel development) relative to fully expanded are the main source of nitrogen for the developing kerflag leaf. We have identified highly expressed gene sets, nel, and a major portion of this may be contributed by which reflect the roles of lemma/palea in protecting and the lemma/palea. In addition, being an outer cover, the nourishing the developing kernel. lemma/palea may protect florets and kernels from attack by pathogens and insects. Very little study has been MATERIALS AND METHODS devoted to genes pertaining to the unique functions of these organs. Analysis of genes highly or uniquely Plant Material Barley plants (‘Morex’) were grown in a greenhouse maintained at 16 to 21 C. Plants received supplemental lighting T. Abebe and H.F. Kaeppler, Department of Agronomy, University of Wisconsin, Madison, WI 53706; R.W. Skadsen, USDA-ARS, Cereal from sodium arc vapor lights for 16 h per day. For total RNA Crops Research Unit, 501 Walnut St., Madison, WI 53726. This work isolation, mature lemma/palea, and 4th, 5th, 6th, and flag was supported by the North American Barley Genome Mapping Projleaves were collected. Lemma/palea were collected from ect, the American Malting Barley Association, and the United States spikes at three stages of kernel development: elongating (beDepartment of Agriculture. Received 18 June 2003.* Corresponding tween pollination and milky stages), gelatinous and early author ([email protected]). dough (Skadsen et al., 2000). To avoid variations in gene expression because of differences in development, care was Published in Crop Sci. 44:942–950 (2004). taken to collect leaves with similar stages of development to  Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA the lemma/palea.