Transcription factor as signaling regulatory tools for improving growth processes-Case studies

1. Topic
on
“Transcription factor as signaling regulatory tools for improving growth
processes-Case studies:
Tb1-Lateral branch development, shi4 –Grain shattering, GA1-Dwarfing.
MADS,KNOX-Flowering development, HAT 4-Shade development, AP 2-
EREBP-Biotic/Abiotic stresses.
”
Department of Plant Physiology

2. Tb1 – Lateral branches development
Doebley et.al, 1997
• Teonsinte branched 1 gene as major contribution to this evolutionary
change in maize.
• Tb1 acts as both to repress the growth of axillary organ and to enable
the formation of female inflorescences.
• It increases apical dominance, inflorescence development and sex
determination.
• It Act both to repress the growth of axillary organ & unable the
formation of female inflorescences.

3. Plant material and methods
 The W22 line carrying Tb1 + Teosinte was constructed as
previously described except with six generation of backcrossing.
 To isolation of tb1 gene we used the Mutator (Mu) transposable
element system.
 Homozygous tb1-ref plants were crossed to an active Mu stock and
26000 F1 plants were grown.
 Among these, three new tb1 mutants (tb1,2,3- mum3)were
observed.
 Each new mutant was crossed to maize inbreed A632 and 14
progeny from each of these crossed were grown to small seedlings
and then used for DNA extraction.

4. CONT….
 Using Southern blot analysis with Mu element probes and
there by identified Mu3 element that with tb1- mum1.
 Northern blot analysis with RNA from various tissue and
both mutant and wild type plants revealed a 1.5 kb message
in wild type maize axillary inflorescence, immature internode
and husk.

6. Shi 4 – Grain/Seed shattering
• Seed shattering is an important agricultural trait in crop
domestication.
• SH4 (for grain shattering quantitative trait locus on chromosome 4)
and
• qSH1 (for quantitative trait locus of seed shattering on chromosome
1) genes have been identified as required for reduced seed shattering
during rice domestication.
• we identified a seed shattering abortion1 (shat1) mutant in a wild
rice introgression line.
ZHOU et.al 2012

7. Cont…
 The SHAT1 gene, which encodes an APETALA2 transcription
factor, is required for seed shattering through specifying abscission
zone development in rice.
 Genetic analyses revealed that the expression of SHAT1 in AZ was
positively regulated by the tri-helix transcription factor SH4.
 We also identified a frame shift mutant of SH4 that completely
eliminated AZs and showed non-shattering.
 Results suggest a genetic model in which the persistent and
concentrated expression of active SHAT1 and SH4 in the AZ during
early spikelet developmental stages is required for conferring AZ
identification.
 qSH1functioned downstream of SHAT1 and SH4, through
maintaining SHAT1 and SH4 expression in AZ, thus promoting AZ
differentiation.

8.  The cultivated rice sh4-1 allele achieved an ideal balance between
shattering and threshing during rice domestication.
 Demonstrated that this reduced-shattering sh4-1 allele was quickly
fixed in all rice cultivars, with levels of sequence polymorphism
significantly reduced in both indica and japonica cultivars relative
to the wild progenitors.
 Mutations in theSHAT1 gene also occur in the natural population,
we determined the polymorphism of SHAT1 in different rice
landraces.
 Our high-throughput sequencing results, of 614 accessions of
landraces from China and 330 accessions of international varieties
showed 19 SNPs located in the SHAT1 genic region.
 Eight were located in the UTR regions, four in introns, and seven in
exons. Those SNPs generated
 few functional variants, with most cultivated varieties having the
same protein sequence as that in O. rufipogon W1943.
Selection for Shattering Genes in Domestication

9. METHODS
 Mutant Material and Growth Conditions.
 Characterization of Mutant Phenotype.
 Cloning of SHAT1 and SHAT2.
 RNAi Experiment.
 RNA Extraction and Quantitative RT-PCR Analysis.
 Nuclear Localization Analysis and Transactivation Assay.
 Construction of the SHAT1 Promoter-GUS Fusion and GUS
Assay.
 In Situ Hybridization.

11. GA 1 - Dwarfing
• Gibberellin insensitive dwarf 1 gene.
• In this study, we selected dwarf apple rootstock ‘SH6’ and its cross
parents as materials to clone the GA receptor gene GID1c.
• We conducted GA3 treatment and overexpression of GID1c in
tissue culture seedlings of ‘SH6’.
• Results showed that the expression of GID1c and biosynthesis genes
increased and promoted the accumulation of hormone contents,
which ultimately regulates the growth of ‘SH6’ dwarf apple rootstock
seedlings.
• It also promote germination, control plant height and root growth.
improve tillering, regulate flowering, improve seed production
increasing harvesting index and yield.
Hao et.al,2019

12. Cont..
 In modern apple breeding, the application of dwarf rootstocks can
reduce vegetative growth and lead to dwarfing phenotype, which
improves the fruit yield and quality.
 Aim to analyze the regulation mechanism of GID1c on plant
architecture and dwarfism characteristics in ‘SH6’ dwarf apple
rootstocks and promote its application value in orchards.
 In endogenous hormone regulatory pathways, GA signals and the
GA receptor GID1 may perform key functions that regulate plant
height and architecture.

14. MADS-flowering development
• MADS-box gene family members in response to drought, salt, cold,
heat, and oxidative stress conditions in different developmental
processes of several plants.
• They are key components of gene regulatory networks involved in
plant responses to stress and plant developmental plasticity in
response to seasonal changes in environmental conditions.
• MADS-domain proteins are able to bind DNA as homo or
heterodimers together with other MADS-domain proteins or with
other proteins as part of different protein complexes and function as
tetramers in order to regulate transcription of their target genes.
• MADS-box genes are involved in abiotic stress response in different
developmental processes in several plants
MUNOX et.al, 2019

16. Cont..
 A. OsMADS26 is a negative regulator of drought stress tolerance in rice. The
cartoon represents down regulation of OsMADS26 (OsMADS26-RNAi) and
wild type plants in conditions of water stress for 18 days followed by 15 days
of re-watering.
 B. SlMBP11 is required in order to give more tolerance for salt stress to
tomato plants. Plants are more affected by salt stress condition (100 mM
NaCl) when this gene is down regulated (SIMBP11-RNAi).
 C. Overexpression (OE) of TaMADS51 in transgenic tobacco plants improves
plant growth under phosphorous (P)-deprivation.
 In Arabidopsis,
 D. SVP repress the onset of the flowering in drought escape response at short
day conditions. Cartoons are representative of wild type plants (16 weeks old)
and svp mutants (8 weeks old) subjected to control conditions or drought
regime.
 E.AGL21 is important for LR development in control conditions and under
nitrogen (N)-deprivation.
 F. AGL21 function as a negative regulator of seed germination under osmotic
stress conditions (300 mM mannitol)

17. • Involvement of MADS-Box Genes in Flower
Development.
 Tomato flowers are very sensitive to low temperatures, displaying three
different phenotypes.
 MADS-box genes, including TOMATO APETALA3, are highly
induced under cold stress conditions.
 Stamen specification is regulated by a tetramer integrated by the
MADS-domain proteins AP3, PI, AG, and SEP.
 Tomato develops many different stamen phenotypes under continuous
mild heat conditions (32◦C day, 26◦C night). They include loss of
pollen viability and deformation of some of the anthers into pistil-like
structures.
 The homeotic transformation of the anthers suggested that the
expression patterns of B and C class genes from tomato should be
analyzed two AP3 genes, TAP3 and TOMATO MADS BOX GENE6
(TM6), two PI genes, Le-PISTILLATA (LePI) and TOMATO
PISTILLATA, and two C-class genes TOMATO AGAMOUS1 and
TAG-LIKE1.

18. KNOX- Flowering development
• kn1 and related knotted-like homeobox (knox) genes encode atypical
homeodomain.proteins that have three extra amino acids between helix 1 and helix 2.
• TALE (three amino acid loop extension) family of homeodomains. Two major groups
of plant proteins are found in the TALE class, KNOX and BEL.
• SHOOTMERISTEMLESS (STM).
• knox genes encode homeodomain-containing transcription factors that are required for
meristem maintenance and proper patterning of organ initiation.
• KNOX proteins function as heterodimers with other homeodomains in the TALE
superclass.
• The gibberellin and lignin biosynthetic pathways are known to be negatively regulated
by KNOX proteins, which results in indeterminate cell fates.
• knox genes are not expressed in the P0 region of the meristem and in simple leaves,
expression remains off HAKE et.al, 2004

20. • KNOX gene expression patterns in model monocot and
dicot.
• The maize kn1 and the Arabidopsis STM gene are
expressed throughout shoot meristems.
• Except the P0 cells where the next leaf will initiate.
• BP from Arabidopsis and rs1 from maize are expressed in
subdomains of the shoot apical meristem.

21. HAT 4 – Shade development
• It is member of class II homeodomain-leucine zipper (HD-ZIPII)
transcription factor family have been shown to play an instrumental role in
the responses to shade.
• We recently showed that REVOLUTA (REV), a member of the HD-ZIPIII
family, directly and positively regulates the expression of several genes
involved in shade-induced growth, such as those encoding HDZIPII factors
HAT2, HAT3, ATHB2/HAT4 and ATHB4, and of the components of the
auxin biosynthesis pathway YUCCA5 and TAA1.
• Directly & positively regulates expression of several genes involved in shade
induced growth.
• Main factor - responsible for R: F R ratio. TORRENT et.al, 2012

23. • Our analysis of REV target genes revealed several HD-ZIPII
transcription factors that are directly and positively regulated by
REV.
• Some HD-ZIPII transcription factors are known to be involved in
shade signaling10 and our recent analysis showed that HD-ZIPIIIs
are also involved in shade growth.

24. AP2-EREBP – Biotic/Abiotic stresses
• Plants being sessile in nature have to face several adverse environmental
conditions throughout their life cycle.
• Plant responses to these stresses are complex and involve numerous
physiological, molecular, and cellular adaptations.
• AP2/EREBP family also regulates diverse processes of plant development
and metabolism such as vegetative and reproductive development, cell
proliferation, secondary metabolism and responses to different plant
hormones.
SAXENA et,al., 2018