TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals

Abstract

The molecular genetics of vernalization, defined as the promotion of flowering by cold treatment, is still poorly understood in cereals. To better understand this mechanism, we cloned and characterized a gene that we named TaVRT-1 (wheat [Triticum aestivum] vegetative to reproductive transition-1). Molecular and sequence analyses indicated that this gene encodes a protein homologous to the MADS-box family of transcription factors that comprises certain flowering control proteins in Arabidopsis. Mapping studies have localized this gene to the Vrn-1 regions on the long arms of homeologous group 5 chromosomes, regions that are associated with vernalization and freezing tolerance (FT) in wheat. The level of expression of TaVRT-1 is positively associated with the vernalization response and transition from vegetative to reproductive phase and is negatively associated with the accumulation of COR genes and degree of FT. Comparisons among different wheat genotypes, near-isogenic lines, and cereal species, which differ in their vernalization response and FT, indicated that the gene is inducible only in those species that require vernalization, whereas it is constitutively expressed in spring habit genotypes. In addition, experiments using both the photoperiod-sensitive barley (Hordeum vulgare cv Dicktoo) and short or long day de-acclimated wheat revealed that the expression of TaVRT-1 is also regulated by photoperiod. These expression studies indicate that photoperiod and vernalization may regulate this gene through separate pathways. We suggest that TaVRT-1 is a key developmental gene in the regulatory pathway that controls the transition from the vegetative to reproductive phase in cereals.

Figure 1.

Structure and sequence alignment of TaVRT-1 with other MADS-box members of the API/SQUA family. Barley (Hordeum vulgare) HvBM5 (Schmitz et al., 2000), Lolium temulentum LtMADS1 (Gocal et al., 2001), indica rice OsMADS14 (Monn et al., 1999), japonica rice OsRAP1B (Kyozuka et al., 2000), maize (Zea mays) ZmMADS from EST BE511439, petunia (Petunia hybrida) PhTBP26 (Immink et al., 1999), Arabidopsis AtFUL/AGL8 (Mandel and Yanofsky, 1995), Snapdragon (Antirrhinum majus) AmDEFH28 (Müller et al., 2001), and Arabidopsis AtAP1 (Mandel et al., 1992). Identical and similar amino acids are shaded in black and gray, respectively. MADS-box domain, DNA-binding domain; I, intervening region; K, keratin-like domain; C, C-terminal region. *, Residues identified as being part of a nuclear targeting signal by PSORT (Nakai and Kanehisa, 1992; http://psort.nibb.ac.jp). Ser stretch, NetPhos 2.0 (Blom et al., 1999) predicts phosphorylation sites in this region (http://www.cbs.dtu.dk/services/NetPhos/).

Figure 2.

Mapping of TaVRT-1. a, Schematic representation of the deletion lines used to map the TaVRT-1 gene on chromosomes 5A and 5D. The numbers to the left indicate the deletion breakpoints of each line, where the distal portion of the chromosome is missing. Black boxes represent telomeric C-band markers. Orange boxes indicate the regions containing Vrn-1 and TaVRT-1. b, DNA gel-blot analysis of genomic DNA from wheat cv Chinese Spring (CS) and chromosome 5A and 5D deletion lines.

Figure 3.

Expression of TaVRT-1 in several wheat cultivars and other cereals. Unvernalized (control) plants were grown for 12 d at 20°C, whereas vernalized plants were grown for 45 d at 4°C after 7 d at 20°C. RNA from two spring wheat (cvs Glenlea and Manitou), one spring barley (cv Winchester), four winter wheat (cvs Absolvent, Monopole, Fredrick, and Norstar), and a winter rye (cv Musketeer) were analyzed. The blot was hybridized with the specific TaVRT-1 probe. rRNA is shown as a load control.

Figure 4.

Expression of TaVRT-1 in relation to vernalization, COR gene expression, FLN, and development of FT. RNA gel-blot analyses showing TaVRT-1 transcript accumulation in wheat parental lines spring wheat cv Manitou (a) and winter wheat cv Norstar (b) and in the wheat near-isogenic lines winter wheat cv Manitou (c) and spring wheat cv Norstar (d). Blots were hybridized sequentially with the specific TaVRT-1 probe and the WCS120 and WCS19/LEA3-L2 probes. rRNA is shown as a load control. FLN (—) and LT₅₀ (....) for wheat cv Manitou (e) and wheat cv Norstar (f), and their NILs are also illustrated.

Figure 5.

Expression of TaVRT-1 during phenological development in wheat. Spring wheat cv Manitou and winter wheat cv Norstar were grown for 14 d at 20°C under LD photoperiod and were then vernalized at 4°C for the times indicated. Shoot apices were dissected and analyzed for the appearance of a double-ridge structure. Arrow indicates the double-ridge formation indicative of transition to the reproductive phase. RNA gel blots indicating TaVRT-1 transcript level are shown for each time point.

Figure 6.

Expression of a barley TaVRT-1 homolog in vernalization-insensitive barley cv Dicktoo during cold acclimation. Barley plants were grown at 4°C for 0 to 98 d under SD and LD. Northern analysis was as described in Figure 3.

Figure 7.

Expression of a barley, TaVRT-1 homolog, during phenological development in barley cv Dicktoo under different photoperiods. Barley plants were grown under SD and LD photoperiods for 70 d of cold acclimation. Apical shoot development in 0-, 49-, and 70-d-old plants is presented. Arrow indicates the double-ridge formation indicative of transition to the reproductive phase. RNA gel blots indicating transcript level are shown for each time point.

Figure 8.

TaVRT-1 accumulation and phenological development in wheat during de-acclimation under different photoperiods. Wheat cv Norstar plants were acclimated for 56 d at 4°C under SD and LD photoperiods and then de-acclimated at 20°C for 14 d (56 + 14) under the same photoperiod. In the LD 56+14 treatment, the shoot apex has advanced development beyond the double-ridge phase. RNA gel blots indicating TaVRT-1 transcript level are shown for each time point.