SummaryA chimeric mitochondrial DNA (mtDNA) configuration of the cytoplasmic male-sterile (cms) sorghum line ISl112C includes a 321 bp open reading frame designated orfl07, encoding a predicted polypeptide product of 11.85 kDa. The open reading frame, similar to several other genes associated with cms, consists of amino-terminal sequences derived from an obligate gene. Unlike other examples to date, however, the carboxy-terminal sequences are highly similar to the carboxy terminus of an open reading frame implicated in cms of rice, orf79. The amino-terminal 31 residues of orfl07 are 84% similar to atp9, and the carboxy-terminal 49 residues are 57% identical and 80% similar to the carboxy terminus of orf79. Transcripts of off107 are edited, with four C-to-U changes that alter amino acids. Sorghum lines partially or fully restored to fertility exhibit a high-efficiency internal-orfl07 transcript processing activity, precluding abundant wholelength transcripts, while male-sterile lines exhibit only a trace of the activity. Previous data on the abundance of a 12 kDa in organello-synthesized polypeptide in male-sterile versus male-fertile lines are correlated with differential o#107 transcript processing activity of these lines. Examinations of backcross and F 2 lines suggest a gametophytic mode of restoration, and indicate that enhanced transcript processing activity is necessary, but not sufficient, to restore full fertility. These novel observations indicate that mitochondrial open reading frames associated with cms

Sequencing of sorghum mitochondrial atp6 cDNA clones revealed 19 C-to-U transcript editing events within a 756 bp-conserved core gene; three were silent and 16 resulted in 15 amino acid changes. Only one edit, which was silent, was found in the 381 bp amino-extension to the core gene. Eleven of the 15 changed amino acids were identical with or else represented conservative changes compared to yeast atp6. Editing of a CAA codon to TAA truncates the carboxy-terminus to a position identical to that of yeast. The frequency of editing at sites which change amino acids was very high in contrast to partial editing at silent, third base, sites.

During pollen maturation, single-celled microspores undergo a rapid phase of starch biosynthesis, presumably from sucrose stored in the vacuoles. Very little is known about the genes and the intracellular controls that regulate the sucrose → starch pathway in such microspores. We show here RNA profiles in sorghum microspores during the transition from vacuolated microspore to immature pollen at two specific stages of pollen development: early, showing little or no detectable starch, and late, when pollen are active in starch-filling. We have also examined two near-isogenic cytoplasmic male sterile lines that retain fully turgid immature pollen until the starch-filling stage, but remain starch-deficient, and ultimately form inviable pollen. Using maize cDNA clones, both temporal and genotypic differences were observed in the expression profiles of four metabolic genes (soluble or vacuolar invertase, Ivr2; sucrose synthase, Sus1; phosphoglucomutase, Pgm; and a subunit of ADPG pyrophosphorylase, Bt2), two similar metabolic regulatory genes (Grf1 and Grf2, encoding 14-3-3 proteins) and a transcription factor, ZmMADS1. Western blot analyses were conducted to visualize sucrose synthase, SS2; AGPase, BT2 and the GRF (14-3-3) proteins. Temporal differences of both a qualitative and quantitative nature were observed in all three proteins in both male-fertile and male-sterile lines. We suggest that these changes in gene expression may result from increased and decreased sink strengths and associated changes in carbohydrate metabolism in starch-filled (fertile) and starch-deficient (sterile) pollen, respectively.

Insect pests cause significant global agricultural damage and lead to major financial and environmental costs. Crops contain intrinsic defenses to protect themselves from such pests, including a wide array of specialized secondary metabolite-based defense chemicals. These chemicals can be induced upon attack (phytoalexins) or are constitutive (phytoanticipins), and can have a direct impact on the pests or be used indirectly to attract their natural enemies. They form part of a global arms race between the crops and their insect pests, with the insects developing methods of suppression, avoidance, detoxification, or even capture of their hosts defensive chemicals. Harnessing and optimizing the chemical defense capabilities of crops has the potential to aid in the continuing struggle to enhance or improve agricultural pest management. Such strategies include breeding for the restoration of defense chemicals from ancestral varieties, or cross-species transfer of defense metabolite production.

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC(3)F(1) population. Three AFLP markers linked to rf4were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC(1)F(1) individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated.

We describe fundamental characteristics of sorghum mitochondrial orf25, urf209, and a related chimeric configuration, orf265/130, which is restricted to the IS1112C source of cytoplasmic male sterility in sorghum. Transcripts of urf209 are edited at ten nucleotides, resulting in nine amino-acid changes predicted from genomic sequences. The cDNA-predicted polypeptide product is 23.6 kDa, while Western blot analyses identify a product of 20k Da. Transcription of urf209 is characterized by one or two transcripts, dependent on nuclear background, but this difference is not related to male fertility status. The orf265/130 chimeric region includes 288 bp 95% identical to sequences 5' to maize T-cytoplasm T-urf13 and atp6, which includes a common transcription initiation site, and terminates with a recombinational event involving urf209. The urf209 similarity extends 189 bp, followed by sequences duplicated 5' to sorghum atp6-2. Sequences immediately 3' to the atp6-2 similarity include a second in-frame start codon, defining orf130. Structural features 5' to orf130 are shared with motifs found 5' to several translated mitochondrial open reading frames. The orf265/orf130 configuration is uniquely transcribed, and transcripts of orf130 exhibit one silent RNA editing event. Transcription in somatic cells is not altered by male fertility status.

Male-sterile sorghum carrying the IS1112C cytoplasm represents an unusual example of aberrant microgametogenesis wherein microspores develop into inviable pollen that remain physically intact until anther exsertion. These inviable pollen grains do not deposit starch, yet fluoresce with the vital stain fluoroscein diacetate. We sought to elucidate the extent of differential gene expression in this subtle example of defective microgametogenesis through cDNA-AFLP transcript profiling of near-isogenic male-fertile and male-sterile plants at an early stage representing early-mid microspores to early pollen, 7-11 days prior to anthesis, and a late stage representing young to nearly mature pollen, spanning the terminal 96 h of pollen development. The transition from early to late stages is characterized by changes in abundance of nearly 33% of shared transcripts, and early-or late-specific expression of about 10% of transcripts. Malesterile plants exhibit extensive changes in regula-tory patterns characteristic of fertile plants, including premature expression of late-specific, and prolonged expression of early-specific, transcripts. Genome-wide transcriptome patterns indicate the expression of an estimated 12,000 genes in early-mid microspores, and the abundance of at least 15% of these transcripts is altered in male-sterile plants. A near-isogenic line restored to male fertility is characterized by apparent normalized expression of most of these transcripts. The development of the microgametophyte is thus characterized by dynamic programmed changes in gene expression, and the expression of male sterility compounds these changes in a complex manner.

No abstract