Rongbin Hu

Water-Deficit Inducible Expression of a Cytokinin Biosynthetic Gene IPT Improves Drought Tolerance in Cotton

Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in water-limited regions. One approach to engineer drought tolerance is by delaying drought-induced senescence via up-regulation of cytokinin biosynthesis. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis, under the control of a water-deficit responsive and maturation specific promoter PSARK was introduced into cotton and the performance of the PSARK::IPT transgenic cotton plants was analyzed in the greenhouse and growth chamber conditions. The data indicate that PSARK::IPT-transgenic cotton plants displayed delayed senescence under water deficit conditions in the greenhouse. These plants produced more root and shoot biomass, dropped fewer flowers, maintained higher chlorophyll content, and higher photosynthetic rates under reduced irrigation conditions in comparison to wild-type and segregated non-transgenic lines. Furthermore, PSARK::IPT-transgenic cotton plants grown in growth chamber condition also displayed greater drought tolerance. These results indicate that water-deficit induced expression of an isopentenyltransferase gene in cotton could significantly improve drought tolerance.

TAP46 Plays a Positive Role in the ABSCISIC ACID INSENSITIVE5-Regulated Gene Expression in Arabidopsis

A protein phosphatase 2A-associated protein, regulates ABA-regulated gene expression in Arabidopsis. TAP46 is a protein phosphatase2A (PP2A)-associated protein that regulates PP2A activity in Arabidopsis (Arabidopsis thaliana). To study how PP2A is involved in abscisic acid (ABA) signaling in plants, we studied the function of TAP46 in ABA-regulated seed maturation and seedling development. Expression of TAP46 coincides with the action of ABA in developing seeds and during seed germination, and the TAP46 transcript reaches to the highest level in mature seeds. Real-time polymerase chain reaction analysis indicates that external ABA can increase TAP46 transcript level transiently during seed germination. Overexpression of TAP46 increases plant sensitivity to ABA, while tap46 knockdown mutants are less sensitive to ABA during seed germination, suggesting that TAP46 functions positively in ABA signaling. Overexpression of TAP46 also leads to lower PP2A activity, while tap46-1 knockdown mutant displays higher PP2A activity, suggesting that TAP46 negatively regulates PP2A activity in Arabidopsis. Both TAP46 and PP2A interact with the ABA-regulated transcription factor ABA INSENSITIVE5 (ABI5) in vivo, and TAP46’s binding to ABI5 can stabilize ABI5. Furthermore, TAP46’s binding to the phosphorylated ABI5 may prevent PP2A or PP2A-like protein phosphatases from removing the phosphate from ABI5, thereby maintaining ABI5 in its active form. Overexpression of TAP46 and inhibition of activities of PP2A or PP2A-like protein phosphatases can increase transcript levels of several ABI5-regulated genes, suggesting that TAP46 is a positive factor in the ABA-regulated gene expression in Arabidopsis.

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H+-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na+/H+-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.

Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 of PP2A was studied using both loss-of-function and gain-of-function analyses. Loss-of-function mutant pp2a-c5-1 displayed more impaired growth during root and shoot development, whereas overexpression of PP2A-C5 conferred better root and shoot growth under different salt treatments, indicating that PP2A-C5 plays an important role in plant growth under salt conditions. Double knockout mutants of pp2a-c5-1 and salt overly sensitive (sos) mutants sos1-1, sos2-2 or sos3-1 showed additive sensitivity to NaCl, indicating that PP2A-C5 functions in a pathway different from the SOS signalling pathway. Using yeast two-hybrid analysis, four vacuolar membrane chloride channel (CLC) proteins, AtCLCa, AtCLCb, AtCLCc and AtCLCg, were found to interact with PP2A-C5. Moreover, overexpression of AtCLCc leads to increased salt tolerance and Cl- accumulation in transgenic Arabidopsis plants. These data indicate that PP2A-C5-mediated better growth under salt conditions might involve up-regulation of CLC activities on vacuolar membranes and that PP2A-C5 could be used for improving salt tolerance in crops.

Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR Is Essential for PROTEIN PHOSPHATASE 2A Holoenzyme Assembly and Plays Important Roles in Hormone Signaling, Salt Stress Response, and Plant Development

An Arabidopsis phosphotyrosyl phosphatase activator is essential for assembly of a protein phosphatase and plays important roles in hormone signaling, salt stress response, and plant development. PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation.

The E3 ligase AtCHIP positively regulates Clp proteolytic subunit homeostasis

The caseinolytic peptidase (Clp) core proteins are essential for plant growth and development, especially for chloroplast function. Antisense or overexpression of ClpP4, which is one of the Clp core subunits, causes chlorotic phenotypes in Arabidopsis. An E3 ligase gene, AtCHIP, has previously been found to ubiquitylate ClpP4 in vitro. ClpP4 antisense and overexpressing plants that also overexpressed AtCHIP were constructed to explore the effect of AtCHIP on ClpP4. Overexpression of AtCHIP was found to rescue the chlorotic phenotypes of both ClpP4 antisense and overexpressing plants. The unbalanced levels of Clp core proteins in ClpP4 antisense and overexpressing plants with overexpression of AtCHIP were similar to wild-type levels, suggesting that AtCHIP regulates Clp core proteins. The results also show that AtCHIP can interact with ClpP3 and ClpP5 in yeast and ubiquitylate ClpP3 and ClpP5 in vitro. This suggests that AtCHIP is directly related to ClpP3 and ClpP5. Given these results, the inference is that through selective degradation of Clp subunits, AtCHIP could positively regulate homeostasis of Clp proteolytic subunits and maximize the production of functional chloroplasts. Similar results were obtained from transgenic tobacco plants, suggesting that regulation of the Clp protease by AtCHIP is conserved.

The yield difference between wild-type cotton and transgenic cotton that expresses IPT depends on when water-deficit stress is applied

Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in transgenic plants. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis is one of them. Interestingly, when IPT-transgenic cotton was field-tested at two different sites, Texas and Arizona, different results were obtained. To explain this phenomenon, reduced irrigation experiments with different timing in applying water deficit stress were conducted. It was found that the timing of water deficit stress is critical for IPT-transgenic cotton to display its yield advantage over control plants (i.e. wild-type and segregated non-transgenic plants). If water deficit stress occurs before flowering (vegetative phase), IPT-transgenic cotton would outperform control plants; however, if water deficit stress occurs at or after flowering (reproductive phase), there would not be a yield difference between IPT-transgenic and control cotton plants. This result suggests that an early induction of IPT expression (before first flowering) is needed in order to realize the benefits of IPT-expression in transgenic plants that face water-deficit stress later in development.

Overexpression of the PP2A-C5 gene confers increased salt tolerance in Arabidopsis thaliana

ABSTRACT Protein phosphatase 2A (PP2A) was shown to play important roles in biotic and abiotic stress signaling pathways in plants. PP2A is made of 3 subunits: a scaffolding subunit A, a regulatory subunit B, and a catalytic subunit C. It is believed that the B subunit recognizes specific substrates and the C subunit directly acts on the selected substrates, whereas the A subunit brings a B subunit and a C subunit together to form a specific PP2A holoenzyme. Because there are multiple isoforms for each PP2A subunit, there could be hundreds of novel PP2A holoenzymes in plants. For an example, there are 3 A subunits, 17 B subunits, and 5 C subunits in Arabidopsis, which could form 255 different PP2A holoenzymes. Understanding the roles of these PP2A holoenzymes in various signaling pathways is a challenging task. In a recent study,1 we discovered that PP2A-C5, the catalytic subunit 5 of PP2A, plays an important role in salt tolerance in Arabidopsis. We found that a knockout mutant of PP2A-C5 (i.e. pp2a-c5–1) was very sensitive to salt treatments, whereas PP2A-C5-overexpressing plants were more tolerant to salt stresses. Genetic analyses between pp2a-c5–1 and Salt-Overly-Sensitive (SOS) mutants indicated that PP2A-C5 does not function in the same pathway as SOS genes. Using yeast 2-hybrid analysis, we found that PP2A-C5 interacts with several vacuolar membrane bound chloride channel proteins. We hypothesize that these vacuolar chloride channel proteins might be PP2A-C5s substrates in vivo, and the action of PP2A-C5 on these channel proteins could increase or activate their activities, thereby result in accumulation of the chloride and sodium contents in vacuoles, leading to increased salt tolerance in plants.

Morphological, Physiological and Proteomic Analyses Provide Insights into the Improvement of Castor Bean Productivity of a Dwarf Variety in Comparing with a High-Stalk Variety

Ricinus communis displays a broad range of phenotypic diversity in size, with dwarf, common, and large-sized varieties. To better understand the differences in plant productivity between a high-stalk variety and a dwarf variety under normal growth conditions, we carried out a comparative proteomic study between Zhebi 100 (a high stalk variety) and Zhebi 26 (a dwarf variety) combined with agronomic and physiological analyses. Over 1000 proteins were detected, 38 of which differed significantly between the two varieties and were identified by mass spectrometry. Compared with Zhebi 100, we found that photosynthesis, energy, and protein biosynthesis related proteins decreased in abundance in Zhebi 26. The lower yield of the dwarf castor is likely related to its lower photosynthetic rate, therefore we hypothesize that the lower yield of the dwarf castor, in comparing to high stalk castor, could be increased by increasing planting density. Consequently, we demonstrated that at the higher planting density in Zhebi 26 (36,000 seedlings/hm2) can achieve a higher yield than that of Zhebi 100 (12,000 seedlings/hm2). Proteomic and physiological studies showed that for developing dwarf R. communis cultivar that is suitable for large scale-production (i.e., mechanical harvesting), it is imperative to identify the optimum planting density that will contribute to higher leaf area index, higher photosynthesis, and eventually higher productivity.