Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants

BACKGROUND Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialling systems as they are described in the literature that our experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays. RESULTS The drivers for the development of this hydroponic system were: 1) the exclusion of light from the growth solution; 2) to simplify the handling of individual plants, and 3) the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution. Following germination, the liquid growth media was exchanged with the experimental solution, and after 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micropipette techniques such as electrophysiology or SiCSA. CONCLUSIONS We present an optimised plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present ‘tips and tricks’ for the easy adoption of this hydroponic culture system.Simon J Conn, Bradleigh Hocking, Maclin Dayod, Bo Xu, Asmini Athman, Sam Henderson, Lucy Aukett, Vanessa Conn, Monique K Shearer, Sigfredo Fuentes, Stephen D Tyerman and Matthew Gilliha

Calcium storage in plants and the implications for calcium biofortification

Calcium (Ca) is an essential nutrient for plants and animals, with key structural and signalling roles, and its deficiency in plants can result in poor biotic and abiotic stress tolerance, reduced crop quality and yield. Likewise, low Ca intake in humans has been linked to various diseases (e.g. rickets, osteoporosis, hypertension and colorectal cancer) which can threaten quality of life and have major economic costs. Biofortification of various food crops with Ca has been suggested as a good method to enhance human intake of Ca and is advocated as an economically and environmentally advantageous strategy. Efforts to enhance Ca content of crops via transgenic means have had promising results. Overall Ca content of transgenic plants has been increased but in some cases adverse affects on plant function have been observed. This suggests that a better understanding of how Ca ions (Ca²⁺) are stored and transported through plants is required to maximise the effectiveness of future approaches.Maclin Dayod, Stephen Donald Tyerman, Roger Allen Leigh and Matthew Gilliha

Phenetic Analysis of Cultivated Black Pepper (Piper nigrum L.) in Malaysia

Phenetic analysis of all the black pepper cultivars in Malaysia is crucial to determine the morphological difference among them. The objective of this study is to ascertain the morphological distinctness and interrelationships among the cultivars to ensure registration of each variety under the Plant Variety Protection Act, as a prerequisite toward implementation of a monovarietal farm policy in the future. Cluster analysis revealed that cultivars “Semongok Aman” and “Semongok 1” have high distinctness values for identification; thus, varietal diagnosis for the two cultivars is easy. Cultivars “Nyerigai,” “India,” “Semongok Perak,” and “Semongok Emas” were grouped in the most diverse clusters among the ten cultivars studied. The four cultivars have a similarity index as high as 92%; however, investigation of leaf width, leaf width-length ratio, seed weight, and conversion rate (fresh to black pepper) gives the ability to determine the characteristic differences. Cultivars “Lampung Daun Lebar” and “Yong Petai” have a similarity of 96%; however, the two showed distinctive differences in leaf width, leaf length-width ratio, spike thickness, and spike length characteristics. On the contrary, cultivars “Kuching” and “Sarikei” showed the highest similarity index, at 98%, and thus are among the most difficult cultivars to diagnose the morphological difference. However, the principle component analysis showed that the fruit size and seed diameter were the important diagnostic key characteristics. Overall, the leaf width, leaf width-length ratio, fruit spike, and conversion rate characteristics are among the key characteristics to differentiate among cultivars of black pepper in Malaysia. At the same time, the principle component analysis carried out has enlightened some interrelationships on the morphological characteristics between cultivars. This information is crucial for the future of the plant varietal improvement program in Malaysia

Calcium delivery and storage in plant leaves: exploring the link with water flow

Calcium (Ca) is a unique macronutrient with diverse but fundamental physiological roles in plant structure and signalling. In the majority of crops the largest proportion of long-distance calcium ion (Ca2+) transport through plant tissues has been demonstrated to follow apoplastic pathways, although this paradigm is being increasingly challenged. Similarly, under certain conditions, apoplastic pathways can dominate the proportion of water flow through plants. Therefore, tissue Ca supply is often found to be tightly linked to transpiration. Once Ca is deposited in vacuoles it is rarely redistributed, which results in highly transpiring organs amassing large concentrations of Ca ([Ca]). Meanwhile, the nutritional flow of Ca2+ must be regulated so it does not interfere with signalling events. However, water flow through plants is itself regulated by Ca2+, both in the apoplast via effects on cell wall structure and stomatal aperture, and within the symplast via Ca2+-mediated gating of aquaporins which regulates flow across membranes. In this review, an integrated model of water and Ca2+ movement through plants is developed and how this affects [Ca] distribution and water flow within tissues is discussed, with particular emphasis on the role of aquaporins.Matthew Gilliham, Maclin Dayod, Bradleigh J. Hocking, Bo Xu, Simon J. Conn, Brent N. Kaiser, Roger A. Leigh and Stephen D. Tyerma

Identification of Quantitative Trait Loci Controlling High Calcium Response in <i>Arabidopsis thaliana</i>

<div><p>Natural variation for primary root growth response to high Ca stress in <i>Arabidopsis thaliana</i> was studied by screening a series of accessions (ecotypes) under high Calcium (40 mM CaCl₂ ) conditions. The genetic basis of this variation was further investigated by QTL analysis using recombinant inbred lines from Landsberg <i>erecta</i> (L<i>er</i>)×Cape Verde Islands (Cvi) cross. Four QTLs were identified in chromosome 1, 2 and 5,and named response to high Calcium (<i>RHCA</i>) 1–4. The three QTLs (<i>RHCA1</i>, <i>RHCA2</i> and <i>RHCA4</i>) were further confirmed by analysis of near isogenic lines harboring Cvi introgression fragments in L<i>er</i> background. Real-time PCR analysis showed that several genes associated with high Ca response including <i>SMT1</i> and <i>XHT25</i> have changed expression pattern between L<i>er</i> and near isogenic lines. These results were useful for detecting molecular mechanisms of plants for high Ca adaption.</p></div