Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT.

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.

Regulation of flowering in the long-day grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T gene.

Seasonal control of flowering often involves leaf sensing of daylength coupled to time measurement and generation and transport of florigenic signals to the shoot apex. We show that transmitted signals in the grass Lolium temulentum may include gibberellins (GAs) and the FLOWERING LOCUS T (FT) gene. Within 2 h of starting a florally inductive long day (LD), expression of a 20-oxidase GA biosynthetic gene increases in the leaf; its product, GA(20), then increases 5.7-fold versus short day; its substrate, GA(19), decreases equivalently; and a bioactive product, GA(5), increases 4-fold. A link between flowering, LD, GAs, and GA biosynthesis is shown in three ways: (1) applied GA(19) became florigenic on exposure to LD; (2) expression of LtGA20ox1, an important GA biosynthetic gene, increased in a florally effective LD involving incandescent lamps, but not with noninductive fluorescent lamps; and (3) paclobutrazol, an inhibitor of an early step of GA biosynthesis, blocked flowering, but only if applied before the LD. Expression studies of a 2-oxidase catabolic gene showed no changes favoring a GA increase. Thus, the early LD increase in leaf GA(5) biosynthesis, coupled with subsequent doubling in GA(5) content at the shoot apex, provides a substantial trail of evidence for GA(5) as a LD florigen. LD signaling may also involve transport of FT mRNA or protein because expression of LtFT and LtCONSTANS increased rapidly, substantially (>80-fold for FT), and independently of GA. However, because a LD from fluorescent lamps induced LtFT expression but not flowering, the nature of the light response of FT requires clarification.

Photoperiodic regulation of flowering in perennial ryegrass involving a CONSTANS -like homolog.

Photoperiod and vernalization are the two key environmental factors of the floral induction of perennial ryegrass (Lolium perenne L.). Transition from vegetative to reproductive growth will only occur after an extended vernalization period, followed by an increase in day length and temperature. Here we report on the isolation and characterization of a L. perenne gene (LpCO ) that is homologous to CONSTANS , and which is tightly coupled to the floral inductive long day signal. Like other monocot CO-like proteins, the LpCO contains a zinc finger domain with a non-conserved B-Box2. Although the B-Box2 has been demonstrated to be essential for the function of the Arabidopsis CO (AtCO), LpCO is able to complement the Arabidopsis co-2 mutant, and ectopic expression in Arabidopsis wild type leads to early flowering. The LpCO transcript exhibits diurnal oscillations and is expressed at higher levels during long days.

MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth.

In contrast to well-studied dicot plants like Arabidopsis and Antirrhinum, relatively few genes controlling the transition to flowering and flower development of agronomically important monocot species have been identified. In perennial ryegrass (Lolium perenne) the transition from vegetative to reproductive growth is triggered by an obligate vernalization period (primary induction) of at least 12 weeks at temperatures below 5 degrees C under short days, followed by increased temperature and day length (secondary induction). Here we report the isolation of nine ryegrass MADS-box (LpMADS) genes by a differential display method specific to this family of transcription factors. Three of the nine MADS-box genes show homology to the APETALA 1 (AP1) subfamily, two to the SEPALLATA (SEP) subfamily, one to the AGAMOUS-LIKE 6 (AGL6) subfamily, and three show homology to the newly identified OsMADS1 subfamily. The three AP1 homologues are up-regulated, both in the shoot apex and in leaves, in response to vernalization, while expression of the other six are increased by secondary induction during inflorescence development, although not in leaves. Differences in the sequence and hierarchy of flowering gene expression patterns indicate that the Arabidopsis-based flowering model is not completely applicable to explain the molecular events leading to the floral transition in grasses.

Do 14-3-3 proteins and plasma membrane H+-AtPases interact in the barley epidermis in response to the barley powdery mildew fungus?

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHAI), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H+-ATPase. These effects are seen specifically in the inoculated epidermis and not in the whole leaf. We propose that 14-3-3 proteins are involved in an epidermis-specific response to the powdery mildew fungus, possibly via an activation of the plasma membrane H+-ATPase.