Understanding the role of de novo tracheary elements in Nicotiana interfamily grafting

概要

報告番号

※

第

主

論文題目
氏

名

号

論

文

の

要

旨

Understanding the role of de novo tracheary elements
in Nicotiana interfamily grafting（ タ バ コ 属 植 物 の
異科接木における道管形成の分子機構と機能の研究）
黄 朝 琨 （ HUANG Chaokun）

論 文 内 容 の 要 旨
Plant grafting has been widely used since antiquity for crop improvement.
Historical records have revealed that ancient Chinese and Greeks utilized it
since 1560 B.C. Nicotiana benthamiana ( Nb ) have recently been evidenced, which
can graft onto diverse angiosperms and establish successful interfamily grafts.
In the study of chapter Ⅰ , we established two interfamily graft combinations,

Nicotiana benthamiana grafted onto the inflorescence stem of Arabidopsis
( Nb / At ) and Glycine max grafted on Arabidopsis ( Gm / At ), proposing a standard of
successful

interfamily

grafting

is

achieved

reproductive

growth

in

scion.

Physiological anatomy of the graft union revealed the N. benthamiana has strong
ability in xylem formation during grafting. De novo tracheary elements (TEs)
were differentiated in the callus at the graft boundary which determining scion
growth

post

interfamily

grafting.

The

2,3,5-triiodobenzonic

acid

(TIBA)

treatment inhibited de novo TE formation during N. benthamiana grafting,
further limiting the scion growth. Moreover, we found the early limitation on de

novo TEs affecting the scion growth at late phase.
VASCULAR-RELATED NAC-DOMAIN7 ( VND7 ) is a transcription factor
gene that plays a crucial role in xylem differentiation. The ectopic expression of

VND7 induces the formation of secondary cell walls with spiral patterns in
multiple types of plant cells. In the study of chapter Ⅱ , we identified four
homologs of VND7 in Nicotiana benthamiana , and assigned them the name
NbVND7-1 to NbVND7-4. Promoter activities of NbVND7 genes were detected in
related GUS-fusion transgenic lines. All NbVND7s were expressed in callus
tissues at the graft junction, while NbVND7-1 and NbVND7-2 were more

concentrated on xylem. A novel β-Estradiol inducible gene expression system was
established for ectopic over-expressing NbVND7-2 in plant tissues. Under the
β-Estradiol treatment, NbVND7-2 induced ectopic xylem differentiation in N.

benthamiana seedlings and graft sites. Moreover, β-Estradiol was found
enhancing the post growth of scion in Nb / At interfamily grafts, whereas
excessively induced xylem cells differentiation in graft union causing the scion
dead. This study provides the evidence for suitable application of VND7 in plant
genetic modification.
Grafting is a plant propagation technique widely used in agriculture. A
recent discovery of the capability of interfamily grafting in Nicotiana has
expanded the potential combinations of grafting. In the study of chapter Ⅲ , we
showed that xylem connection is essential for the achievement of interfamily
grafting and investigated the molecular basis of xylem formation at the graft
junction. Transcriptome analyses revealed the expression pattern of xylem
formation related genes in Nicotiana benthamiana ( Nb ) and Glycine max ( Gm )
during interfamily grafting. The up-regulation of Nicotiana benthamiana XYLEM

CYSTEINE PROTEASE ( NbXCP ) genes was validated in tracheary element (TE)
formation. Promoter activities of NbXCP1 and NbXCP2 genes were found in
differentiating TE cells in the stem and callus tissues at the graft junction.
Analysis of a Nbxcp1;Nbxcp2 loss-of-function mutant indicated that NbXCPs
control the timing of de novo TE formation at the graft junction. Moreover, grafts
of the NbXCP1 overexpressor increased the scion growth rate as well as the fruit
size. These results indicate that xylem formation is important for graft viability
and can be a target to improve grafting technique.
Xylem plays a crucial role in transporting water and nutrients from the
roots to the rest of the plant. Mobile metabolites are organic molecules that can
move freely within a plant's vascular system, allowing them to be transported to
different parts of the plant. In the study of chapter Ⅳ , we established graft
combinations using Solanaceae plants, Nicotiana tabacum ( Nt ) and Solanum

lycopersicum ( Sl ). The tissues and xylem sap were collected from intact Nt and Sl
plants,

and

Nt / Sl ,

Sl / Nt

Chromatography-Quadrupole

Time

grafted
of

plants.

Flight-tandem

We
Mass

used

Liquid

Spectrometry

(LC-QTOF-MS/MS) to reveal hundreds of metabolites which were transported in
the xylem during grafting. Moreover, the analysis of retention time in mass
spectrometry (RT) and mass-to-charge ratio (m/z) identified as the same
substance in plant tissue and sap.