Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S . tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes ( AtCBF1-3 ) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 °C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.     

Molecular control of cold acclimation in trees

Frost tolerance is an acquired characteristic of plants that is induced in response to environmental cues preceding the onset of freezing temperatures and activation of a cold acclimation program. In addition to transient acclimation to low non-freezing temperatures and enhancing survival to short frost episodes during the growth season, perennial woody plants need additionally to survive the cold winter months. Trees have evolved a complex dynamic process controlling the development of dormancy and freezing tolerance that secures accurate initiation and termination of the overwintering process. Although the phenology of overwintering has been known for decades, only recently has there been progress in elucidating the molecular mechanisms of dormancy and freezing tolerance development in perennial plants. Current molecular and genomic studies indicate that herbaceous annual and woody perennial plants share similar cold acclimation mechanisms. Both the signal processes controlling cold acclimation and the cold-regulated target genes appear to be shared by herbaceous and woody plants. However, the dormancy development during overwintering brings new players in the molecular control of seasonal cold acclimation of woody perennials.     

Characterization of MORE AXILLARY GROWTH Genes in Populus

Background

Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants.

Methodology/Principal Finding

Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants.

Conclusion/Significance

This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.     

Relationship between photosynthetic rate,water use and leaf structure in desert annual and perennial forbs differing in their growth

Specific leaf area (SLA) is a key trait to screen plants for ecological performance and productivity; however, the relationship between SLA and photosynthesis is not always up-scalable to growth when comparing multiple species with different life cycles. We explored leaf anatomy in annual and perennial species of Physaria, and related it to photosynthesis and water loss. The annual Physaria gracilis had higher SLA, thinner leaves, and lower investment in protective tissues, than perennial P. mendocina. Physaria angustifolia (annual), and P. pinetorum (perennial) showed intermediate values. Both perennials had a thicker palisade and high photosynthesis, relative to annuals. The larger leaf veins of perennials should allow high water availability to the mesophyll. The thicker palisade should determine high resistance to water flow and help explain their high water-use efficiency. These leaf functions reflect the construction of long-lived leaves that efficiently use resources under environmental limitations of arid environments.

     

Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato

We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii . Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2 °C in S. tuberosum and up to 4 °C in S. commersonii . Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii , AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBF pathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species.     

Partitioning of Carbohydrates in Annual and Perennial Cotton (Gossypium hirsutum L.)

An analysis of the partitioning of carbohydrates in annual andperennial cotton was made to ascertain the distribution of assimilatesand constitution of reserves. Root/shoot dry matter ratio ishigh in perennial cotton and this plant shows a preferentialaccumulation of dry matter in roots corresponding to its adaptationto drought. Starch content is also higher in perennial cottonroots than in annual. It can be said that the earlier maturingthe cultivar, the lower the root/shoot ratio and the lower thestarch content. Nevertheless, at the whole plant level in annualcotton the starch content is highest in leaves where it is accumulatedbefore migration, and stem wood, and lowest in root and bark.While starch content in roots of annuals declines after 3 months,it is still increasing in perennials. Accumulation of carbohydratesas reserve material can be modified by selection and such selectionis accompanied by an increase in the activities of ß-amylasein exporting organs: leaves, woody tissue of the stem, and barkbut not in roots. Invertase activities were highest in leavesbut did not respond to selection. Non-irrigated cotton had ahigher activity of ß-amylase in leaves and stem woodcorresponding to the mobilization of reserve assimilates. Smallerincreases were observed in the activity of invertase. High yieldingannual cottons show a higher activity of ß-amylaseand invertase in leaves corresponding to a higher capacity ofassimilate transfer. Also a comparison was made from emergenceto 4 months of the partitioning of carbohydrates between leaf,stem and roots in annual and perennial cotton. In conclusionperennial cotton apparently owes its drought resistance to apartitioning of assimilates that favours the growth of the rootsystem and the accumulation of starch reserves in roots. Key words: Gossypium hirsutum L, carbohydrates, partitioning     

The role of CBF transcriptional activators in two Citrus species (Poncirus and Citrus) with contrasting levels of freezing tolerance

Citrus species are considered cold sensitive, yet Poncirus trifoliata , an interfertile Citrus relative, is extremely cold tolerant and can survive freezes at −20°C when fully cold acclimated. To better understand the molecular basis of this difference in freezing tolerance, C-repeat-binding factors (CBFs) from P. trifoliata and Citrus paradisi (grapefruit) were isolated and characterized. Based on our results, P. trifoliata and C. paradisi do contain a CBF pathway for cold-regulated gene expression, and similar to other species, they both appear to contain small CBF families. The nucleotide and amino acid sequences of PtCBF1 and CpCBF1 are highly homologous. However, differences in cold-induced expression were observed. PtCBF1 accumulates both earlier and to higher levels than CpCBF1 . Furthermore, CORc115 , a cold-induced group II LEA gene, also accumulates earlier and to higher levels in P. trifoliata in response to cold temperatures. The regulatory region of CORc115 contains a putative C-repeat/dehydration-responsive element that is specifically recognized and bound by PtCBF, indicating that CORc115 is a target of PtCBF1. This study provides the first evidence of a correlation between CBF expression (timing and quantity) and the degree of cold tolerance in two closely related species with wide differences in cold tolerance. The importance of using Poncirus and Citrus , two non-model organisms, to address new questions about the CBF pathway is illustrated, as these results provide novel evidence that the CBF expression pattern may play an important role in the notable difference in cold tolerance between these two related species.     

miRNA control of vegetative phase change in trees

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Glutamine, arginine and the amino acid transporter Pt-CAT11 play important roles during senescence in poplar

Background and Aims

Nitrogen (N) availability in the forest soil is extremely low and N economy has a special importance in woody plants that are able to cope with seasonal periods of growth and development over many years. Here we report on the analysis of amino acid pools and expression of key genes in the perennial species Populus trichocarpa during autumn senescence.

Methods

Amino acid pools were measured throughout senescence. Expression analysis of arginine synthesis genes and cationic amino acid transporter (CAT) genes during senescence was performed. Heterologous expression in yeast mutants was performed to study Pt-CAT11 function in detail.

Key Results

Analysis of amino acid pools showed an increase of glutamine in leaves and an accumulation of arginine in stems during senescence. Expression of arginine biosynthesis genes suggests that arginine was preferentially synthesized from glutamine in perennial tissues. Pt-CAT11 expression increased in senescing leaves and functional characterization demonstrated that Pt-CAT11 transports glutamine.

Conclusions

The present study established a relationship between glutamine synthesized in leaves and arginine synthesized in stems during senescence, arginine being accumulated as an N storage compound in perennial tissues such as stems. In this context, Pt-CAT11 may have a key role in N remobilization during senescence in poplar, by facilitating glutamine loading into phloem vessels.     

Leaf temperature of desert sand dune plants: perspectives on the adaptability of leaf morphology

The leaf temperature of six annual and six perennial plant species was monitored during spring and summer on a sand dune ecosystem in the delta Mediterranean coast of Egypt. During winter, leaves of all tested perennial species attained temperatures higher than the air temperature at night and shortly after sunrise, with maximum leaf–air temperature differences reaching up to 8°C. The lowest differences were less than 1°C. Around noon, the leaves of several species attained temperatures lower than that of the air whereas others showed higher temperatures. The opposite was true during summer, when leaf temperatures were lower than air temperature. The maximum leaf–air temperature differences occurred after midnight towards sunrise and reached up to 10°C. The lowest differences were found around noon and were of less than 5°C. The annual plant species have more pronounced variations than perennials in their leaf temperatures during the night and for most of the day. The leaves were heated or cooled a few degrees above or below the air temperature. The results are discussed in relation to the morphological characters of the leaves. The variation in leaf temperature at different times of the day was significantly related to leaf morphology, specific leaf area, thickness, volume, leaf area index and the surrounding environment.