39.768 Plant Molecular Genetics
Lecture 18, part 2 of 3

C. Hormone-regulated genes

1. A group of SAUR RNA genes is clustered in soybean genome.

McClure, B.A., Hagen, G., Brown, C.S., Gee, M.A. and Guilfoyle, T.J. (1989) Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean. The Plant Cell vol. 1, 229-239.

ORGANIZATION OF THE SAUR LOCUS [Fig. 2]

5 SAUR genes (presumably generated by gene duplication) are clustered at 1.25kb intervals at one locus. Three highly-conserved sequence domains were identified: two adjacent elements (DUE and NDE) are located upstream from the transcription start site, while a third (DST) is located in the 3'-untranslated region of the mRNA transcript. These sites are presumptive regulatory sequences.

The SAUR genes are a good example of a multigene family that has arisen through gene duplication.

2. Auxin-mediated gene-expression is tissue-specific.

Used gene-specific cDNA probes to examine differential transcription of SAUR genes in slot blots of cDNAs.

Isolated nuclei from soybean elongating hypocotyl sections (EHS) were incubated with ³²P-dATP. RNA was isolated from nuclei and hybridized with cDNA-containing slot blots.

TRANSCRIPTIONAL REGULATION OF THE SAUR GENES [Fig. 1]

A - Genes 10A, 6 and 15 all show induction by 2,4D
B - 6B is strongly induced in EHS, but not in leaf. GH3 is strongly induced in both.
C - Maximal transcriptional activation of 6B occurrs within 10 min of 2,4D addition.

3. Auxin induces expression of SAUR RNAs during gravitrophism

McClure, B.A. and Guilfoyle, T. (1989) Rapid redistribution of auxin-regulated RNAs during gravitropism. Science 243:91-93.

Gravitropism - bending of plants in response to gravity.

Hypothesis: auxins facilitate gravitropism by modulating the rate of cell extension. Specifically, cells on lower survace elongate more than cells on upper surface.

MCLURE & GUILFOILE [Fig 1]

EHS - elongating hypocotyl sections
Probes:

SAUR - small auxin up RNA (mixed probe of 4 SAUR's used in northern: 6B, 6G, 10A5 & 15C)
5A - non-auxin-inducible clone

RNAs:
C - control
T - 2,4D

This RNA gel blot shows that none of the SAUR mRNAs are expressed in soybean EHS in the absence of auxin. Auxin is required for their expression in EHS.

MCLURE & GUILFOILLE [Fig. 2]

a) Tissue-printing using ³⁵S-antisense probes
Seedlings are cut longitudinally, exposing tissue. Tissue is pressed onto a nitrocellulose filter, and RNA from the cells binds to the filter according to the location of cells. The filter is hybridized with radioactive probe to visualize the location of cells expressing the corresponding gene.
b) vertical seedlings - symmetrical distribution of SAUR expression around the major axis of hypocotyl

horizontal seedlings -
10 min.: symmetric
20min. : decrease in SAUR on upper right, increase in SAUR expression on lower surface. where cell extension is greatest

45min.: strongest skewing of SAUR to lower surface

180min. apices have returned to vertical, SAUR's are symmetrical again.

Note: auxin-insensitive transcript 5A remains symmetrically distributed

c) Results:

i) SAUR induction occurs before onset of gravitropism
ii) Distribution of SAUR coincides with site of rapid cell extension

iii) SAUR's expressed in cortex & epidermal layers of hypocotyl.

SAUR mRNAs are induced prior to auxin-mediated gravitropic response on lower surfaces of soybean seedlings. Distribution of SAUR transcripts coincides with sites of rapid cell elongation.

III. SIGNAL TRANSDUCTION.

A. Hormone-regulated DNA binding proteins

1. Detection of DNA binding proteins by gel-shift assays.

2. 5' region of α-amylase gene in rice has GA3-specific DNA binding domain.

Ou-Lee, T.-M., Turgeon, R. and Wu, R. (1988) Interaction of giberellin-induced factor with the upstream region of an -amylase gene in rice aleurone tissue.

During germination, embryo secretes giberellin into endosperm, which causes induction of alpha-amylase in aleurone tissue, which forms the outer layer of the endosperm.

a. A 500bp Hind3/Sal1 fragment including the transcription start site is bound by a GA3-induced nuclear protein.

PUTATIVE REGULATORY SEQUENCES OF RICE α-AMYLASE GENE. [Fig. 4]

Nuclear extracts from rice aleurone tissues were incubated with radioactively-labeled HS500 fragment, a 500bp fragment from the promoter and translation initiation region of the rice alpha-amylase gene

ALPHA-AMYLASE DNA BINDING ACTIVITY WITH RICE ALEURONE PROTEIN. [Fig. 2]

Competitor: unlabeled HS500 DNA

BINDING ACTIVITY OF -AMYLASE DNA IN RICE TISSUES [Ou-Lee & Wu, Fig. 5]
R - roots

L - leaves

GA+D deembryonated half-seeds, 2days GA₃

GA+A rice aleurone, 2days GA₃

D deembryonated half-seeds, no giberellin

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