Plants in Microgravity: Molecular and Technological Perspectives.

Plants are vital components of our ecosystem for a balanced life here on Earth, as a source of both food and oxygen for survival. Recent space exploration has extended the field of plant biology, allowing for future studies on life support farming on distant planets. This exploration will utilize life support technologies for long-term human space flights and settlements. Such longer space missions will depend on the supply of clean air, food, and proper waste management. The ubiquitous force of gravity is known to impact plant growth and development. Despite this, we still have limited knowledge about how plants can sense and adapt to microgravity in space. Thus, the ability of plants to survive in microgravity in space settings becomes an intriguing topic to be investigated in detail. The new knowledge could be applied to provide food for astronaut missions to space and could also teach us more about how plants can adapt to unique environments. Here, we briefly review and discuss the current knowledge about plant gravity-sensing mechanisms and the experimental possibilities to research microgravity-effects on plants either on the Earth or in orbit.

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis.

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O2•-) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O2•- (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed.

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana.

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses.

Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis.

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.

Functional Analysis of the Arabidopsis thaliana CDPK-Related Kinase Family: AtCRK1 Regulates Responses to Continuous Light.

The Calcium-Dependent Protein Kinase (CDPK)-Related Kinase family (CRKs) consists of eight members in Arabidopsis. Recently, AtCRK5 was shown to play a direct role in the regulation of root gravitropic response involving polar auxin transport (PAT). However, limited information is available about the function of the other AtCRK genes. Here, we report a comparative analysis of the Arabidopsis CRK genes, including transcription regulation, intracellular localization, and biological function. AtCRK transcripts were detectable in all organs tested and a considerable variation in transcript levels was detected among them. Most AtCRK proteins localized at the plasma membrane as revealed by microscopic analysis of 35S::cCRK-GFP (Green Fluorescence Protein) expressing plants or protoplasts. Interestingly, 35S::cCRK1-GFP and 35S::cCRK7-GFP had a dual localization pattern which was associated with plasma membrane and endomembrane structures, as well. Analysis of T-DNA insertion mutants revealed that AtCRK genes are important for root growth and control of gravitropic responses in roots and hypocotyls. While Atcrk mutants were indistinguishable from wild type plants in short days, Atcrk1-1 mutant had serious growth defects under continuous illumination. Semi-dwarf phenotype of Atcrk1-1 was accompanied with chlorophyll depletion, disturbed photosynthesis, accumulation of singlet oxygen, and enhanced cell death in photosynthetic tissues. AtCRK1 is therefore important to maintain cellular homeostasis during continuous illumination.