Basi molecolari e biochimiche del carattere sofficità nella cariosside del genere Avena
Author(s)
Gazza, Laura
Date Issued
February 26, 2010
Type
Doctoral Thesis
Abstract
Amongst the cereal crops, Avena sativa (hexaploid species with genome AACCDD),
is characterized by an extremely soft endosperm texture with mean SKCS values around - 20,
much lower than those measured in common wheat (14 to 90), rye and barley (40 to 60) and
einkorn wheat (-5 to 0). This high softness causes some negative agronomic and technological
aspects. The analysis of genetic, molecular and biochemical basis of soft texture of oats grain
is the subject of the present PhD project. Kernel texture in common wheat is mainly
controlled by puroindolines A (Pin-A) and B (Pin-B), which accumulate on starch granules in
the endosperm. The observation that A. sativa contains DNA sequences similar to genes
coding for wheat puroindolines, suggested that proteins (avenoindolines) encoded by these
DNA sequences could play a role in determining kernel texture in oats. Therefore, proteins
associated with starch granules in endosperm of hexaploid, tetraploid and diploid genotypes
of Avena were analyzed by mono and two-dimensional electrophoresis, protein sequencing and immunochemical techniques (Western blotting). PCR amplification, RT-PCR, RACE and
gene sequencing were also used to identify and characterize the genes coding for starch-bound
proteins in those oats species. Evidence has been obtained that oats is the only cereal species
with soft kernels in which puroindoline-like proteins (avenoindolines) do not play a
significant role in the modulation of kernel texture, as suggested by the fact that
avenoindolines occur in trace amounts in Avena sativa, whereas they are absent in all diploid
and tetraploid Avena species analyzed. In oats species, the role of determinant factors of
kernel texture seems to be played by proteins, called here vromindolines (vromi is the greek
name for oats), which are characteristic of this species and absent in wheat. Vromindolines
(Vin) were found to belong to the 2S protein family. They proved to be similar to
puroindolines in their aminoacid sequences, number (10) and position of cysteine residues,
basic isoelectric point, molecular weight (13-14 KDa) and the ability to accumulate on starch
granules. However, vromindolines exhibit some peculiar characteristics such as a central
domain with four tryptophan residues (instead of five or three typical of Pin-A and Pin-B,
respectively), the presence of several charged amino acids at both ends of the mature protein,
and insolubility in chloroform/methanol mixtures.
When fractionated by acid electrophoresis (A-PAGE), vromindolines in diploid, tetraploid
and hexaploid Avena species split into two families called Vin-A and Vin-B, each consisting
of three major components (Vin-A1, Vin-A2, Vin-A3, Vin-B1, Vin-B2 and Vin-B3).
Vromindolines Vin-B1, Vin-B2 and Vin-B3 are encoded by Vinb-1, Vinb-2 and Vinb-3 genes
each present in two or three copies. Vin-A proteins are synthesized from 7 days post-anthesis
(DPA), while Vin-B proteins are synthesized from 14 DPA. Accumulation of both Vin-A and
Vin-B on starch granules continues for more than 35 DPA. Results also indicated that the
amount of Vin-A and Vin-B accumulated on starch granules is particularly high, about the
1.5% of the dry weight of the kernel, at least 10 times greater than that observed for
puroindolines in wheat.
PCR amplifications with three primer pairs specific for genes coding for Vin-B1, Vin-
B2 and Vin-B3 did not produce amplicons in barley, rye, common wheat, durum wheat and
T. timopheevii. On the contrary, Am -genome T. monococcum ssp monococcum and hexaploid
wheat T. zhukovskyi with AmAuG genomes were found to possess the Vinb-2Xa gene, suggesting a phylogenetic relationship between genome Am and the A genome in Avena species.
Biochemical and genetic characterization of proteins associated to starch granules in A.
sativa led to identification of a gene coding for a novel, highly hydrophobic protein, of 150
aminoacids, here called Avena-α Amilase-Trypsin–Inhibitor (AATI).
In order to demonstrate the softening effect of vromindolines on oats kernel texture,
durum wheat cv. Svevo (mean SKCS value >90 ) was transformed by the biolistic method
using a pair of plasmids containing either Vinb-2Xa or Vinb-3Xa. Twenty-four T1 plants
expressing both transgenes were grown in plant growth chambers, and characterized for their
genetic structure and kernel texture. The frequencies of the two phenotypic classes for the
presence/absence of both Vinb-2Xa and Vinb-3Xa genes in the T2 kernels obtained from
transgenic durum wheat plants G5 and G30, as determined by PCR amplification of genomic
DNAs with vromindoline-specific primers, were closed to the 3:1 ratio, suggesting that the
two transgenes behaved as a single Mendelian unit. Co-segregation of Vinb-2Xa and Vinb-
3Xa was also observed in the progeny of transgenic plant G29. However, the segregation data for transgenes in the progeny of plant G29 was close to the 15:1 ratio for two independent
Mendelian units. Mean SKCS values lower than 30, which are typical of soft kernels, were
observed in the progeny of several T1 plants of cv. Svevo, suggesting that vromindolines Vin-
B2 and Vin-B3 are responsible of the soft texture of oats kernels and are able to modulate
grain texture in durum wheat as well.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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