cccipk14-gene-function-analysis-to-illuminate-efficient-root

<em>CcCIPK14</em> Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Beijing Forestry University

For non-model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non-transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT-PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15-39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.

Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants.

Pigeonpea is an important economic crop in the world and is mainly distributed in tropical and subtropical regions. In order to further expand the scope of planting, one of the problems that must be solved is the impact of soil acidity on plants in these areas. Based on our previous work, we constructed a time series RNA sequencing (RNA-seq) analysis under aluminum (Al) stress in pigeonpea. Through a comparison analysis, 11,425 genes were found to be differentially expressed among all the time points. After clustering these genes by their expression patterns, 12 clusters were generated. Many important functional pathways were identified by gene ontology (GO) analysis, such as biological regulation, localization, response to stimulus, metabolic process, detoxification, and so on. Further analysis showed that metabolic pathways played an important role in the response of Al stress. Thirteen out of the 23 selected genes related to flavonoids and phenols were downregulated in response to Al stress. In addition, we verified these key genes of flavonoid- and phenol-related metabolism pathways by qRT-PCR. Collectively, our findings not only revealed the regulation mechanism of pigeonpea under Al stress but also provided methodological support for further exploration of plant stress regulation mechanisms.

Time Series RNA-seq in Pigeonpea Revealed the Core Genes in Metabolic Pathways under Aluminum Stress.

The flowers of okra (Abelmoschus esculentus) open and wilt within only a few hours, and this is accompanied by accumulation of hyperoside, a secondary metabolite in the flavonoid pathway. However, little is known about the relationship between flavonoids and flowering. Here, we found that exogenous application of hyperoside extended the duration of the full-blooming period by more than 3-fold, and this was accompanied by a 14.7-fold increase in the expression of CALCIUM-DEPENDENT PROTEIN KINASE6 (AeCDPK6). Gene expression profiling indicated that the transcription factor AeMYB30 was co-expressed with AeCDPK6, and detailed protein interaction and phosphorylation experiments together with yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated an interaction between AeMYB30 and AeCDPK6. AeCDPK6 specifically phosphorylated AeMYB30S191, leading to increased protein stability and prevention of degradation. Furthermore, AeMYB30 directly bound to the promoter of AeUF3GaT1, a key enzyme in the hyperoside biosynthesis pathway. Analysis of transgenic plants showed that AeCDPK6 was required for the hyperoside-induced phosphorylation of AeMYB30 to enhance its stability and transcriptional activity. Ectopic expression of AeCDPK6 promoted hyperoside accumulation and prolonged the full-blooming period in an AeMYB30-dependent manner. Our results indicate the role of AeCDPK6-AeMYB30 in the molecular mechanism by which hyperoside regulates the period of full blooming in okra, a plant with a short duration of flowering.

CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra.

Actin depolymerising factor (ADF) is an actin binding protein that is ubiquitous in animal and plant cells. It plays an important role in plant growth and development, as well as resistance to biotic and abiotic stress. The research of plant ADF family has been restricted to Arabidopsis thaliana (L.) Heynh. and some herb crops, but no woody cash crops have been reported to date. All members of the Cajanus cajan (L.) Millsp. ADF (CcADF) family were identified from the pigeon pea genome, and distributed among the four subfamilies by phylogenetic analysis. CcADFs were relatively conservative in gene structure evolution, protein structure and functional expression, and different CcADFs showed specific expression patterns under different treatments. The expression characteristics of several key CcADFs were revealed by analysing the stress response pattern of CcADFs and the time series RNA-seq of aluminium stress. Among them, CcADF9 in the first subgroup specifically responded to aluminium stress in the roots; CcADF3 in the second subgroup intensively responded to fungal infection in the leaves; and CcADF2 in the fourth subgroup positively responded to various stress treatments in different tissues. This study extended the relationship between plant ADF family and aluminium tolerance, as well as adding to the understanding of CcADF family in woody crops.

Multidimensional analysis of actin depolymerising factor family in pigeon pea under different environmental stress revealed specific response genes in each subgroup.

ATP-binding cassette (ABC) transporters are a class of proteins found in living organisms that mediate transmembrane transport by hydrolyzing ATP. They play a vital role in the physiological processes of growth and development in plants. The most numerous sub-type transporter in the ABC transporter family is the ABCG group and which have the most complex function in a plant's response to abiotic stresses. Our study focused on the effect of ABCG transporters in the adaptation of the pigeon pea to adverse environments (such as drought, salt, temperature, etc.). We conducted a functional analysis of ABCG transporters in the pigeon pea and their role in response to abiotic stresses. A total of 51  genes () were identified, and phylogenetic analysis was conducted. We also identified the physicochemical properties of the encoded proteins, predicted their subcellular localization, and identified of the conserved domains. Expression analysis showed that  genes have different expression profiles with tissues and abiotic stresses. Our results showed that  was up-regulated at low temperatures, and  was up-regulated with drought and aluminum stress. The initial results revealed that ABCG transporters are more effective in the abiotic stress resistance of pigeon peas, which improves our understanding of their application in abiotic stress resistance.

The functional analysis of ABCG transporters in the adaptation of pigeon pea () to abiotic stresses.

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a central role in Ca signalling and promote drought tolerance in plants. The CIPK gene family in pigeon pea (Cajanus cajan L.), a major food crop affected by drought, has not previously been characterised. Here, we identified 28 CIPK genes in the pigeon pea genome. Five CcCIPK genes were strongly upregulated in roots upon drought treatment and were selected for further characterisation. Overexpression of CcCIPK13 and CcCIPK14 increased survival rates by two- to three-fold relative to controls after 14 days of drought. Furthermore, the three major flavonoids, genistin, genistein and apigenin, were significantly upregulated in the same transgenic plants. Using CcCIPK14 as bait, we performed a yeast two-hybrid screen and identified six interactors, including CcCBL1. CcCIPK14 exhibited autophosphorylation and phosphorylation of CcCBL1 in vitro. CcCBL1-overexpressed plants displayed higher survival rates upon drought stress as well as higher expression of flavonoid biosynthetic genes and flavonoid content. CcCIPK14-overexpressed plants in which CcCBL1 transcript levels were reduced by RNA interference had lower survival rates, which indicated CcCBL1 in the same pathway as CcCIPK14. Together, our results demonstrate a role for the CcCIPK14-CcCBL1 complex in drought stress tolerance through the regulation of flavonoid biosynthesis in pigeon pea.

The pigeon pea CcCIPK14-CcCBL1 pair positively modulates drought tolerance by enhancing flavonoid biosynthesis.

Flavonoids are secondary metabolites that play important roles in fruit and vegetable development. Here, we examined the function of hyperoside, a unique flavonoid in okra (Abelmoschus esculentus), known to promote both flowering and seed set. We showed that the exogenous application of hyperoside significantly improved pollen germination rate and pollen tube growth by almost 50%, resulting in a 42.7% increase in the seed set rate. Of several genes induced by the hyperoside treatment, AeUF3GaT1, which encodes an enzyme that catalyzes the last step of hyperoside biosynthesis, was the most strongly induced. The transcription factor AeMYB30 enhanced AeUFG3aT1 transcription by directly binding to the AeUFG3aT1 promoter. We studied the effect of the hyperoside application on the expression of 10 representative genes at four stages of reproductive development, from pollination to seed maturity. We firstly developed an efficient transformation system that uses seeds as explants to study the roles of AeMYB30 and AeUFG3aT1. Overexpression of AeMYB30 or AeUF3GaT1 promoted seed development. Moreover, exogenous application of hyperoside partially restored the aberrant phenotype of AeUF3GaT1 RNA-interference plants. Thus, hyperoside promotes seed set in okra via a pathway involving AeUF3GaT and AeMYB30, and the exogenous application of this flavonoid is a simple method that can be used to improve seed quality and yield in okra.

Hyperoside regulates its own biosynthesis via MYB30 in promoting reproductive development and seed set in okra.

Mature pollen germinates rapidly on the stigma, extending its pollen tube to deliver sperm cells to the ovule for fertilization. The success of this process is an important factor that limits output. The flavonoid content increased significantly during pollen germination and pollen tube growth, which suggests it may play an important role in these processes. However, the specific mechanism of this involvement has been little researched. Our previous research found that hyperoside can prolong the flowering period of Abelmoschus esculentus (okra), but its specific mechanism is still unclear. Therefore, in this study, we focused on the effect of hyperoside in regulating the actin-depolymerizing factor (ADF), which further affects the germination and growth of pollen. We found that hyperoside can prolong the effective pollination period of okra by 2-3-fold and promote the growth of pollen tubes in the style. Then, we used Nicotiana benthamiana cells as a research system and found that hyperoside accelerates the depolymerization of intercellular microfilaments. Hyperoside can promote pollen germination and pollen tube elongation in vitro. Moreover, AeADF1 was identified out of all AeADF genes as being highly expressed in pollen tubes in response to hyperoside. In addition, hyperoside promoted AeADF1-mediated microfilament dissipation according to microfilament severing experiments in vitro. In the pollen tube, the gene expression of AeADF1 was reduced to 1/5 by oligonucleotide transfection. The decrease in the expression level of AeADF1 partially reduced the promoting effect of hyperoside on pollen germination and pollen tube growth. This research provides new research directions for flavonoids in reproductive development.

Article	Year
Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants. Plant biotechnology journal\| PubMed ID: 30803117	2019
Time Series RNA-seq in Pigeonpea Revealed the Core Genes in Metabolic Pathways under Aluminum Stress. Genes\| PubMed ID: 32244575	2020
CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra. Journal of experimental botany\| PubMed ID: 32249299	2020
Multidimensional analysis of actin depolymerising factor family in pigeon pea under different environmental stress revealed specific response genes in each subgroup. Functional plant biology : FPB\| PubMed ID: 32970987	2021
The functional analysis of ABCG transporters in the adaptation of pigeon pea () to abiotic stresses. PeerJ\| PubMed ID: 33552725	2021
The pigeon pea CcCIPK14-CcCBL1 pair positively modulates drought tolerance by enhancing flavonoid biosynthesis. The Plant journal : for cell and molecular biology\| PubMed ID: 33733535	2021
Hyperoside regulates its own biosynthesis via MYB30 in promoting reproductive development and seed set in okra. Plant physiology\| PubMed ID: 33743011	2021
Hyperoside promotes pollen tube growth by regulating the depolymerization effect of actin-depolymerizing factor 1 on microfilaments in okra. Horticulture research\| PubMed ID: 34193835	2021

Hongyan Cao

Biography

JoVE Journal Publications

Other Publications