BackgroundGrape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine.ResultsOver the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified ...

Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcription-PCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficit-treated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition.

A peptide that strongly stimulates the secretion of juvenile hormone from corpora allata in vitro (allatotropin) has been purified from extracts of heads of pharate adult Manduca sexta. The primary structure of this 13-residue peptide has been determined: H-Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH(2). This neurohormone has no sequence similarity with any known neuropeptide from other organisms. Synthetic allatotropin, as well as truncation fragments, including one with the five amino terminal residues deleted, showed in vitro activity indistinguishable from that of native allatotropin.

A peptide (Manduca sexta ailatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-ArgPhe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neurohormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistihable from that of native M. sexta aliatostatn. The EDsO of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was =2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata ofadult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.Juvenile hormone (JH), which is synthesized and released by the retrocerebral corpora allata (CA), plays a vital role in insect development, primarily in the control of metamorphosis, adult sexual maturation, and reproduction (1, 2). Environmental and physiological factors influence neurosecretory centers in the brain that affect the activity of CA through peptidergic materials either transported directly to the CA by axons or released into the blood (3). These neuropeptides may be stimulatory (allatotropins, e.g., refs. 4-8) (27). However, instead of assaying for JH production by chloroform extraction followed by TLC analysis, we used a rapid isooctane partitioning assay for JH (28). In the cross-species studies, we used the same in vitro technique (one or two pair of CA per incubation).Extraction and Preliminary Purification (Steps 14). Thirty thousand trimmed heads of M. sexta (fresh weight, "'.1460 g) were processed in three batches of 10,000. Each batch was defatted by homogenizing in 2 liters of ice-cold acetone and filtered. Residues were extracted with 1.4 liters of 1 M HOAc/20 mM HCO (containing 0.1 mM phenylmethylsulfonyl fluoride and 0.01 mM pepstatin A, prepared immediately before use) and centrifuged at 10,000 x g for 30 min at 40C. The pellet was extracted and centrifuged twice using a total volume of 2.8 liters of the same solution. The combined supernatants were stirred with swollen SP-Sephadex C-25 resin (300 ml) overnight. The resin was allowed to settle for 2 hr, subsequently poured into a Bio-Rad column (50 x 300 mm), and equilibrated with 1 M HOAc. Material was eluted from the column sequentially with 1-liter volumes of 0.05 M NH4OAc (pH 4.0) and 0.05 M, 0.1 M, 0.2 M, 0.4 M, and 0.8 M NH4OAc (pH 7.0). The 0.1 M and 0.2 M fractions, which had M. sexta AS (Mas-AS) activity, were applied directly to 10 g of reversed-phase Vydac C4 packing material (20-30 pAm, contained in a 75-ml polypropylene syr...

At the end of each developmental stage, insects perform the ecdysis sequence, an innate behavior necessary for shedding the old cuticle. Ecdysis triggering hormones (ETHs) initiate these behaviors through direct actions on the CNS. Here, we identify the ETH receptor (ETHR) gene in the moth Manduca sexta, which encodes two subtypes of GPCR (ETHR-A and ETHR-B). Expression of ETHRs in the CNS coincides precisely with acquisition of CNS sensitivity to ETHs and behavioral competence. ETHR-A occurs in diverse networks of neurons, producing both excitatory and inhibitory neuropeptides, which appear to be downstream signals for behavior regulation. These peptides include allatostatins, crustacean cardioactive peptide (CCAP), calcitonin-like diuretic hormone, CRF-like diuretic hormones (DHs) 41 and 30, eclosion hormone, kinins, myoinhibitory peptides (MIPs), neuropeptide F, and short neuropeptide F. In particular, cells L 3,4 in abdominal ganglia coexpress kinins, DH41, and DH30, which together elicit the fictive preecdysis rhythm. Neurons IN704 in abdominal ganglia coexpress CCAP and MIPs, whose joint actions initiate the ecdysis motor program. ETHR-A also is expressed in brain ventromedial cells, whose release of EH increases excitability in CCAP͞MIP neurons. These findings provide insights into how innate, centrally patterned behaviors can be orchestrated via recruitment of peptide cotransmitter neurons.ecdysis sequence ͉ ecdysis triggering hormone ͉ G protein-coupled receptors ͉ neuropeptide ͉ steroid I nnate behaviors are highly stereotypic and fully functional in the absence of prior experience. Examples include nurturing, nest building, courtship, and escape behaviors as well as certain developmental behaviors such as the ecdysis sequence, necessary for the shedding of old cuticle at the end of each developmental stage in insects. Because the ecdysis sequence is hormonally controlled, it provides unique opportunities to define mechanisms by which chemical messengers assemble and regulate the performance of behaviors (for review, see ref. 1).The ecdysis sequence is initiated through direct actions of bloodborne pre-ecdysis triggering hormone (PETH) and ecdysis triggering hormone (ETH) on the CNS (2, 3). These peptides are released from endocrine Inka cells, which are present throughout the Insecta (4-6). In larval Manduca sexta, the sequence consists of three behavioral phases (3). In the first phase, PETH activates preecdysis I behavior; subsequently, ETH activates preecdysis II and ecdysis behaviors. These behaviors can be elicited by ETH from the isolated CNS in vitro and, hence, are centrally patterned (3, 7).The ecdysis behavioral sequence is repeatedly acquired and lost throughout the life history of insects (3,8). The signaling pathways triggered by PETH and ETH are assembled at the end of each developmental stage through steroid-induced gene expression, which is critical for synthesis of PETH and ETH in Inka cells and sensitivity of the CNS to these peptides. Once ecdysis is performed, CNS sensitivity to ET...